Methods and compositions for managing vascular conditions

ABSTRACT

This disclosure relates to methods and compositions for managing vascular conditions by targeting microRNA. In certain embodiments, the disclosure relates to antisense, RNA interference, and blocking oligonucleotide therapeutic compositions and uses related thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/107,936 filed Aug. 21, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/772,978 filed Sep. 4, 2015, which is theNational Stage of International Application No. PCT/US2014/023396 filedMar. 11, 2014, which claims the benefit of priority to U.S. ProvisionalApplication No. 61/776,201 filed Mar. 11, 2013, U.S. ProvisionalApplication No. 61/904,026 filed Nov. 14, 2013, and U.S. ProvisionalApplication No. 61/916,449 filed Dec. 16, 2013. The entirety of each ofthese applications is hereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under HL075209 andHL080711 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 11236USDIV_ST25.txt. The text file is 6 KB, wascreated on Dec. 3, 2019, and is being submitted electronically viaEFS-Web.

BACKGROUND

Atherosclerosis, i.e., hardening of the arteries, occurs when substancessuch as fat and cholesterol build up in the walls of arteries and formplaques. Plaque buildup narrows arteries, also referred to as stenosis,making them stiffer and more difficult for blood to flow. Pieces ofplaque often break off forming an embolism blocking blood flow resultingin tissue damage which is a common cause of heart attacks and stroke.Carotid endarterectomy (CEA) is a surgical procedure used to preventstroke, by correcting stenosis in the common carotid artery. Otherprocedures include endovascular angioplasty and stenting. Surgicalprocedures are expensive and undesirable. In addition, post-operativeincidences of heart attack, stroke, and death are significant. Thus,there is a need for improved methods for treating and preventingatherosclerosis.

Atherosclerosis preferentially occurs in arterial regions exposed todisturbed flow (d-flow), in part, due to alterations in gene expression.Vascular endothelial cells respond to blood flow through mechanosensors,which transduce the mechanical force associated with flow (known asshear stress) into cell signaling events and ultimately bring aboutchanges in gene expression. Disturbed-flow promotes while stable-flowinhibits atherogenesis, respectively, through differential regulation ofpro-atherogenic and atheroprotective genes.

Ni et al. report the discovery of mechanosensitive genes in vivo usingmouse carotid artery endothelium exposed to disturbed flow. Blood, 2010,116(15): e66-73. Boon et al. report MicroRNA-29 in Aortic Dilation. CircRes, 2011; 109:1115-1119. Maegdefessel et al. report inhibition ofmicroRNA-29b reduces murine abdominal aortic aneurysm development. JClin Invest, 2012, 122(2):497-506.

Son et al. report the atypical mechanosensitive microRNA-712 derivedfrom pre-ribosomal RNA by an XRN1-dependent manner induces endothelialinflammation and atherosclerosis. Nature Comm. 2013, 4:3000.

Reference cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to methods and compositions for managingvascular conditions by targeting microRNA. In certain embodiments, thedisclosure relates to antisense, blocking oligonucleotides, and RNAinterference therapeutic compositions and uses related thereto.

In certain embodiments, the disclosure relates to compositionscomprising isolated nucleobase polymers that binds or hybridizes anymiRNA disclosed herein, e.g., human miR-21, miR-133b, miR-378 andmiR-205, for any of the uses disclosed herein.

In certain embodiments, the disclosure relates to composition comprisingan isolated nucleobase polymer that binds RNA of miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACA AUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA. Typically, the nucleobasepolymer is a nucleic acid or nucleic acid mimetic that hybridizes tomiR-205 (SEQ ID NO: 11).

In certain embodiments, the disclosure relates to composition comprisingan isolated nucleobase polymer that binds RNA of miR-663 (SEQ ID NO: 1)CCUUCCGGCGUCCCAGGCGGGGCGCCGCGGGACCGCCCUCGUGUCUGUGGCGGUGGGAUCCCGCGGCCGUGUUUUCCUGGUGGCCCGGCCAUG. Typically, the nucleobasepolymer is a nucleic acid or nucleic acid mimetic that hybridizes tomiR-663 (SEQ ID NO: 1).

In certain embodiments, a nucleobase polymer disclosed herein comprisesmonomers of phosphodiester, phosphorothioate, methylphosphonate,phosphorodiamidate, piperazine phosphorodiamidate, ribose, 2′-O-methyribose, 2′-O-methoxyethyl ribose, 2′-fluororibose, deoxyribose,1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol,P-(2-(hydroxymethyl)morpholino)-N,N-dimethylphosphonamidate,morpholin-2-ylmethanol, (2-(hydroxymethyl)morpholino)(piperazin-1-yl)phosphinate, or peptide nucleic acids or combinationsthereof.

In certain embodiments, a nucleobase polymer disclosed herein is 3′ or5′ terminally conjugated to a polyphosphate, polyphosphate ester,hydrocarbon, polyethylene glycol, saccharide, polysaccharide, cellpenetrating peptide or combinations thereof. Typically, the cellspenetrating peptide is a positively charged peptide, arginine-richpeptide, oligoarginine peptide (7-12), or octa-arginine (R8).

In certain embodiments, a nucleobase polymer disclosed herein comprises8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more nucleobases. Incertain embodiments, a nucleobase polymer disclosed herein comprises 8,9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more continuousnucleobases that hybridize SEQ ID NO:1 or SEQ ID NO:11.

In certain embodiments, a nucleobase polymer disclosed herein is thereverse complement of SEQ ID NO:1 or SEQ ID NO:11 has greater than 60%,70%, 80%, 90%, 95% or more sequence identity over a 20, 30, 40, 50, 60,70, 80, or more nucleobase comparison window.

Typically, a nucleobase polymer is less than 100 or 50 nucleobases.

In certain embodiments, the disclosure relates to particles comprising acyclodextrin polymer or a particle with a lipid, or hydrophilic membraneand ionizable or cationic core comprising a nucleobase polymer disclosedherein.

In certain embodiments, this disclosure relates to pharmaceuticalcompositions comprising the nucleobase polymer disclosed herein or aparticle comprising a nucleobase polymer disclosed herein, and apharmaceutically acceptable excipient.

In certain embodiments, the disclosure relates to methods of treating orpreventing a vascular disease or condition comprising administering aneffective amount of a pharmaceutical composition disclosed herein to asubject in need thereof.

In certain embodiments, the subject is a human that is at risk of,exhibiting symptoms of, or diagnosed with atherosclerosis, aneurysm,peripheral vascular disease, coronary heart disease, heart failure,right ventricular hypertrophy, cardiac dysrhythmia, endocarditis,inflammatory cardiomegaly, myocarditis, vascular heart disease, stroke,cerebrovascular disease, or peripheral arterial disease, or cancer.

In certain embodiments, the subject has type I or type II diabetes,impaired glucose tolerance, elevated serum C-reactive proteinconcentration, vitamin B6 deficiency, dietary iodine deficiency,hypothyroidism, hyperlipidemia, hypertension, or is older than 50 yearsold, or smokes cigarettes daily.

In certain embodiments, the pharmaceutical composition is administeredin combination with a statin, atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,simvastatin, ezetimibe, amlodipine, niacin, aspirin, omega-3 fatty acid,or combinations thereof.

In certain embodiments, the disclosure relates to compositionscomprising a double stranded RNA consisting of between 15 and 30continuous nucleotides of SEQ ID NO:1 or SEQ ID NO:11. In certainembodiments, the double stranded RNA is 3′ end capped with one or morethymidine nucleotides and/or the passenger strand of the RNA comprises5′ end polyphosphate.

In certain embodiments, the disclosure relates to particles comprising alipid membrane and ionizable or cationic core comprising the doublestranded RNA disclosed herein.

In certain embodiments, the disclosure relates to pharmaceuticalcomposition comprising the RNA disclosed herein or a particle comprisingRNA disclosed herein, and a pharmaceutically acceptable excipient.

In certain embodiments, the disclosure relates to methods of treating orpreventing a vascular disease condition comprising administering aneffective amount of a pharmaceutical composition comprising an RNAdisclosed herein or a particle comprising RNA disclosed herein.

In certain embodiments, the disclosure relates to methods for inhibitingor preventing atherosclerosis, aortic aneurysm, rheumatoid arthritis andcancer in human patients by treating them with using an effective amountof a compound that inhibits miR-205/miR-712/miR-663 family, which inturn increases or preserves expression of tissue inhibitor ofmetalloproteinease-3 (TIMP3) and agents that increase or preserve theexpression of XRN1 upregulation, and reversion-inducing cysteine-richprotein with Kazal motifs (RECK), wherein said compound is selected fromthe group consisting of microRNA inhibitors of miR-712/miR-205/miR-663family by using nucleic acid sequences that complement the whole or apart of the miRNA sequence such as the seeding sequence. The nucleicacid inhibitors can be synthesized by using locked nucleic acid(LNA)-based designs.

In certain embodiments, the disclosure relates to methods comprisingadministering an effective amount of a microRNA-712/miR-205/miR-663antagonist (anti-miR-712, anti-miR-205, or anti-miR-663) to the diseasedtissues and cell types.

In certain embodiments, the disclosure relates to medical devices, suchas stents, e.g., mesh tube comprising compositions disclosed herein,e.g., recombinant human TIMP3 and RECK, antisense nucleobase polymers,blocking oligonucleotide, gene therapy vectors, anti-miR-205,anti-miR-712, anti-miR-663 or their derivatives, and RNA interferencetherapeutic compositions.

In certain embodiments, the disclosure relates to vascular ornon-vascular medical device coated or conjugated with the inhibitor ofmiR-205, miR-712, or miR-663. In certain embodiments, the inhibitors arelinked to polymers on the surface of the device. In certain embodiments,the inhibitor is integrated to release with biodegradable polymer. Incertain embodiments, medical device is selected from a stents, pacemaker, guide wire, delivery balloon, catheter, bioresorbable vascularscaffold, embolic protection device. In certain embodiments, theinhibitor is a nucleobase polymer.

In certain embodiments, the disclosure relates to a gene therapy using avector that expresses human TIMP3 or RECK in human cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a scheme of naturally occurring d-flow (lesser curvature,LC) and stable flow (s-flow) regions (greater curvature, GC) in theaortic arch. Also shown is the surgically induced d-flow in partialcarotid ligation model in which three of the four caudal branches of theleft common carotid artery (LCA) are ligated, while the contralateralright common carotid artery (RCA) remains untouched as an internalcontrol.

FIG. 1B shows data on endothelial-enriched total RNAs obtained fromintima of mouse (C57BL/6) left carotid (flow-disturbed LCA) and rightcarotid (contralateral control, RCA) at 48 h post ligation were analyzedby gene array (Illumina Bead Chip). Hierarchical clustering analyses ofmechanosensitive miRNAs found in LCA endothelium compared with that ofRCA are shown as heat maps. The expression levels are continuouslymapped on the color scale provided at the top of the figure. Each columnrepresents a single sample pooled from three different LCAs or RCAs, andeach row represents a single miRNA probe (n=3).

FIG. 1C shows data on validation of miRNA microarray results by qPCR.Quantitative PCR (qPCR), using additional independent RNA samples, wasused to validate the above miRNA array data. Ten miRNAs (five up-, fivedownregulated miRNAs at 48 h post ligation) were selected based on foldchange by flow. The qPCR study validated the microarray results for fiveupregulated (miR-712, -330*, -699, -223 and 770-5p).

FIG. 1D shows data on five downregulated (miR-195, -30c, -29b, -26bandlet-7d) miRNAs at the 48 h time point (n=5 each, data shown asmean±s.e.m.; *P<0.05 as determined by paired t-test).

FIG. 1E shows data. To further validate whether the mechanosensitivemiRNAs that were identified in vivo responded specifically to shearstress, expression of these miRNAs were tested in vitro using iMAECsthat were subjected to LS or OS, mimicking s-flow and d-flow in vitro,respectively. Among the nine different miRNAs examined, seven miRNAswere differentially expressed under OS (n=6 each, data shown asmean±s.e.m.; *P<0.05 as determined by paired t-test). These resultsshowed that miR-712 was the most consistently and robustly upregulatedmiRNA both in vivo and under d-flow conditions in vitro.

FIG. 2A shows data where expression of miR-712 was determined by qPCRusing endothelial-enriched RNA obtained from LCA and RCA followingpartial carotid ligation in C57B16 mouse (0-48 h) (n=4, data shown asmean±s.e.m.; *P<0.05 as determined by paired t-test).

FIG. 2B shows data where expression of pre-miR-712 by d-flow in LCA andRCA endothelium following partial ligation at 24 and 48 h wasquantitated by miScript miRNA qPCR assay (n=8 each; *p<0.05 asdetermined by Student's t-test).

FIG. 2C shows data on expression of mature miR-712.

FIG. 2D shows data were expression of pre-miR-712 was measured bymiScript miRNA qPCR in iMAECs exposed to laminar (LS), oscillatory shear(OS) or static (ST) for 24 h (n=6, data shown as mean±s.e.m.; *p<0.05 asdetermined by Student's t-test).

FIG. 2E shows data on mature miR-712.

FIG. 2F shows data where aortic arch (LC and GC) and LCA and RCAobtained at 2 days post ligation from C57B16 mice were subjected tofluorescence in situ hybridization using DIG-labelled miR-712 probe andanti-DIG antibody, which was detected by tyramide signal amplificationmethod using Cy3 and confocal microscopy, (n=6). DAPI nuclear stain;autofluorescent elastic lamina; arrows indicate cytosolic miR-712expression. scale bars, 20 mm.

FIG. 2G shows the potential structure and processing of pre-ribosomalRNA gene, RN45s, which is composed of 18S, 5.8S and 28S rRNA sequenceswith two intervening spacers ITS1 and ITS2. The sequences matchingmurine miR-712 in ITS2 and its putative human counterpart miR-663 inITS1 are indicated as well.

FIG. 2H shows data where expression of DICER in mouse RCA and LCA (2days post ligation) was determined by qPCR (n=4, data shown asmean±s.e.m.; *p<0.05 as determined by paired ttest).

FIG. 2I shows data for DGCR8.

FIG. 2J shows data for XRN1.

FIG. 2K shows data where XRN1 expression in iMAECs exposed to LS or OSfor 24 h was determined by qPCR (n=3 each, data shown as mean±s.e.m.;*p<0.05 as determined by Student's t-test).

FIG. 2L shows data for HUVECs.

FIG. 2M shows data wheremiR-712 expression was induced by treatingiMAECs with XRN1 siRNA but not by DGCR8 siRNA and DICER1 siRNA (n=3each, data shown as mean±s.e.m.; *p<0.05 as determined by Student'st-test).

FIG. 3A shows TIMP3 as a potential target of miR-712 and its link toputative downstream metalloproteinase targets.

FIG. 3B shows the seed sequence of miR-712 (SEQ ID NO: 18) andcomplementary 30-UTR sequence of TIMP3 (SEQ ID NO: 19).

FIG. 3C shows data where iMAECs transfected with dual luciferasereporter plasmids containing wild-type (WT) or mutant TIMP3-30-UTR weretreated with pre-miR-712 or control pre-miR. Firefly luciferase activity(normalized to control Renilla luciferase) indicating TIMP3 expressionwas determined using Luc-Pair miR Luciferase Assay Kit (n=3 each, datashown as mean±s.e.m.; *p<0.05 as determined by Student's t-test).

FIG. 3D shows data where TIMP3 expression in iMAECs determined by qPCRwas decreased by exposure to OS compared with LS or ST for 24 h (n=6,data shown as mean±s.e.m.; *P<0.05 as determined by Student's t-test).

FIG. 3E shows—TIMP3 expression decreased by OS compared with LS for 24h.

FIG. 3F shows data where treatment with pre-miR-712 (20 nM)downregulated TIMP3 expression under LS condition.

FIG. 3G shows data where anti-miR-712 (400 nM) treatment rescuedOS-induced loss of TIMP3. β-actin was used as an internal loadingcontrol.

FIG. 4A shows data where iMAECs were exposed to static, LS or OS for 24h, and the conditioned media were used for TNFa enzyme-linkedimmunosorbent assay (ELISA). LPS was used as a positive control.

FIG. 4B shows data where iMAECs were transfected with TIMP3 expressionvector (TIMP3-OE), TIMP3 siRNA (150 nM), pre-miR-712 (20 nM),anti-miR-712 (400 nM) and respective controls for 24 h. Cells were thenexposed to static, LS or OS for 24 h, and s-TNFα in conditioned mediawas determined by ELISA. (n=6, data shown as mean±s.e.m.; *p<0.05 asdetermined by one-way analysis of variance (ANOVA).

FIG. 4C shows data where iMAECs were transfected with pre-miR-712 (20nM) or control and leukocyte adhesion assay was performed using 2.5×10⁵fluorescent-labelled J774.4 mouse monocyte cells. Adherent cells werecounted (n=5 independent experiments, data shown as mean±s.e.m.; *p<0.05as determined by one-way ANOVA).

FIG. 4D shows data where iMAECs were transfected with TIMP3-OE or itsGFP vector control (GFP-OE), TIMP3 or scrambled control siRNA (siTIMP3or siScr, 150 nM), pre-miR-712 or control pre-miR (20 nM), pre-miR-712with GFP-OE or TIMP3-OE, and no treatment (control), transfection (mock)or no cell controls for 24 h. Cells were then exposed to static, LS orOS for 24 h, and endothelial permeability was determined using aFITC-dextran-based in vitro vascular permeability kit (Cultrex).Fluorescence signals from FITC-dextran in the lower chamber werequantified and plotted as arbitrary fluorescence units (n=3 each fromtwo independent experiments, data shown as mean±s.e.m.; *p<0.05 asdetermined by one-way ANOVA).

FIG. 5A shows data where TexRed-615-labelled control anti-miR or salinewas injected (s.c.) in C57B16 mice. Carotid arteries were dissected out24 h later, and using Zeiss 510 confocal microscope z-stack images ofthe en face sections were obtained. Images were rendered to threedimensions, bounding box was drawn on the area of interest and scaledcoordinate axes were drawn and fluorescence signals from Red channelwere processed and quantified. Graph shows mean fluorescence intensityof TexRed-615 signals from carotids (n=5, data shown as mean±s.e.m.).

FIG. 5B shows data where following partial carotid ligation and HFD for2 weeks, ApoE^(−/−) mice were injected with ⁶⁴Cu-labelled antimiR-712via tail vein. After 3 h, aortic trees including carotids were preparedand autoradiographed, which was used to quantitate percentage ofinjected dose per gram (n=6, data shown as mean±s.e.m.; *p<0.05 asdetermined by Student's t-test). Scale bar, 1 mm.

FIG. 5C shows date used to determine the optimal dose of anti-miR-712,C57B16 mice were injected with anti-miR-712 daily for 2 days (s.c. at 5,20 or 40 mgkg⁻¹ per day) and followed by partial carotid ligation.Mismatched anti-miR-712 (40 mg kg⁻¹ per day) was used as a control.Endothelial-enriched RNAs were prepared from LCA and RCA obtained at 4days post ligation, and miR-712 expression was determined by qPCRshowing optimal effect at 5 mg kg⁻¹ dose (n=4 each, data shown asmean±s.e.m.; *p<0.05 as determined by paired t-test).

FIG. 5D shows data where ApoE^(−/−) mice were partially ligated and fedHFD for 1 week. RCA and LCA frozen sections obtained from these micewere used for immunofluorescence staining with antibody specific toTIMP3 (scale bar, 20 mm).

FIG. 5F shows data for two weeks using versican fragment peptide DPEAAE(scale bar, 20 mm)

FIG. 5E shows date of in situ zymography using DQ-gelatin to determineMMP activity. As a control for the MMP activity assay, some LCA and RCAsections were incubated with MMP inhibitor 1. DAPI and autofluorescentelastic lamina (L=lumen of the artery). Scale bar, 50 μm.

FIG. 6A shows data where ApoE−/− mice were pretreated twice withanti-miR-712 or mismatched control (5 mg·kg⁻¹ each, s.c.) or saline on 1and 2 days before partial ligation. Mice were then fed a HFD andanti-miR and control treatments were continued (twice a week s.c.) for 2weeks. Aortic trees including the carotids were dissected and examinedby bright field imaging and lesion area was quantified (n=10 each, datashown as mean±s.e.m.; *p<0.05 as determined by one-way analysis ofvariance (ANOVA). Scale bar, 1 mm.

FIG. 6B shows frozen sections prepared from the middle parts of thesearteries, denoted by arrows stained with Oil-Red-O and plaque size wasquantified (n=10 each, data shown as mean±s.e.m.; *p<0.05 as determinedby one-way ANOVA). Scale bar, 200 mm.

FIG. 6C shows representative confocal imaging of frozen sectionsimmunostained with CD45 antibody is shown (n=10). Scale bar, 20 μm. Forchronic study, ApoE^(−/−) mice were fed western diet (without anypartial ligation surgery) and were treated with anti-miR-712 (5 mgkg⁻¹,twice a week, s.c.) or mismatched control for 3 months (n=10 each). (f)Aortic trees were dissected and examined by en face Oil-Red-O stainingand the lesion area was quantified (n=4-6 in each group, data shown asmean±s.e.m.; *p<0.05 as determined by Student's t-test). Scale bar, 2mm.

FIG. 6D shows quantified data as in FIG. 6A.

FIG. 6E shows quantified data as in FIG. 6B.

FIG. 6H shows quantified data as in FIG. 6C.

FIG. 6F shows data where aortic arches were longitudinally sectioned

FIG. 6G shows data where stained with Oil-Red-O and plaque size wasquantified (n=4 each, data shown as mean±s.e.m.; *p<0.05 as determinedby Student's t-test). Scale bar, 2 μm.

FIG. 6I shows quantified data in FIG. 6G.

FIG. 6J shows data for TIMP3 overexpression, ApoE^(−/−) mice wereinjected once with AdTIMP3 (10⁸ p.f.u. per animal, via tail vein) orcontrol virus (RAD60, 10⁸ p.f.u. per animal) 5 days before partialcarotid ligation and high-fat diet for 2 weeks. Aortic trees includingthe carotids were dissected and examined by bright field imaging andlesion area was quantified (n=5, data shown as mean±s.e.m.; *p<0.05 asdetermined by Student's t-test). Scale bar, 1 mm.

FIG. 6K shows quantified data in FIG. 6J.

FIG. 7A illustrates seed sequence similarity where highlighted regionshows the seed sequence match between the murine miR-712 (SEQ ID NO: 18)and murine (SEQ ID NO: 20) and human miR-205 (SEQ ID NO: 21), and thoseshown indicate additional conserved sequences.

FIG. 7B shows putative targets of miR-712 and miR-205 obtained fromTargetScan were compared. Venn diagram depicts the common gene targetsof miR-205 and miR-712.

FIG. 7C shows data where expression of precursor and mature miR-205 iniMAECs were exposed to static, LS or OS for 24 h (n=4; data shown asmeans±s.e.m.; *p<0.05 as determined by Student's t-test).

FIG. 7D shows data where expression of precursor and mature miR-205 inhuman aortic endothelial cells (HAECs) were exposed to static, LS or OSfor 24 h (n=4; data shown as means s.e.m.; *p<0.05 as determined byStudent's t-test).

FIG. 7E shows data where expression of mature miR-205 was determinedusing the RNAs obtained from the endothelial-enriched (intimal region)and the leftover medial and adventitia region (M+A) of the LCA and RCAat 24 and 48 h post partial ligation, respectively.

FIG. 7F shows data where expression of TIMP3 was determined by qPCR iniMAECs transfected with increasing concentrations of pre-miR-205 orcontrol pre-miR compared with vehicle controls (mock) (n=3; data shownas means±s.e.m.; *p<0.05 as determined by Student's t-test).

FIG. 7G shows data for HAECs.

FIG. 8 illustrates a working hypothesis that miR-712 inducesinflammation and atherosclerosis by targeting TIMP3; however, it is notintended that embodiment of this disclosure be limited by any particularmechanism. D-flow stimulates miR-712 expression in endothelium by anXRN1-dependent mechanism. Increased miR-712 stimulates endothelialinflammation, permeability and extracellular matrix (ECM) fragmentationby downregulating TIMP3, which is a critical inhibitor of matrixmetalloproteinases (MMPs and ADAMs). Decreased TIMP3 expression bymiR-712 induces inflammation and atherosclerosis by activating amultitude of metalloproteinases: (1) ADAM17/TACE that releasessoluble-TNFα that may induce local and systemic inflammation; (2) ADAMsthat shed junctional VE-cadherin, increasing permeability thatfacilitates LDL and leukocyte infiltration; (3) ADAMTS leading toversican fragmentation; and (4) MMPs leading to ECM degradation leadingto vessel wall remodeling. In addition, miR-712 expression is alsoincreased in whole blood and vascular smooth muscle cells (VSMCs)suggesting either transfer of miR-712 from endothelium to thesecompartments or increase in its local production in these compartments.Increased miR-712 in VSMCs induces their migration while circulatingmiR-712 may affect blood leukocytes further contributing toatherogenesis.

FIG. 9A shows data where expression of miR-712 was determined by qPCRusing endothelial-enriched RNA from AngII-infused abdominal aorta andleftover RNA (media/adventitia) obtained from AngII-infused C57BL/6mouse (0-72 hr).

FIG. 9B shows data where miR-712 expression was tested in immortalizedmouse aortic endothelial cells (iMAECs) and vascular smooth muscle cells(VSMC) in vitro (0-24 hr). (Data were analyzed using ANOVA followed byTukey's post hoc test, mean±S.E. *p<0.05; n=4).

FIG. 9C shows abdominal aortas of C57BL/6 mice obtained at 2-days postAngII-infusion were subjected to fluorescence in situ hybridizationusing digoxigenin-labeled miR-712 probe and confocal microscopy, (n=4).DAPI nuclear stain; Arrows indicate cytosolic miR-712.

FIG. 10A shows data where TIMP3 expression were determined by qPCR iniMAECs treated with AngII (100 ng/ml) and pre-miR-712 (20 nM) with orwithout anti-miR-712 (400 nM) (n=4; p<0.05).

FIG. 10B shows data for RECK.

FIG. 10C shows data where endothelial-enriched RNAs were prepared fromsuprarenal artery of AngII (1 μg/kg/min)-infused mice, which wereinjected with mis-matched control or anti-miR-712 (5 mg/kg; injectedsubcutaneously, s.c.) twice (one and two days before AngII infusion).miR-712, expression was determined by qPCR. (n=4; p<0.05).

FIG. 10D shows data for TIMP3.

FIG. 10E shows data for RECK.

FIG. 10F shows data where iMAECs transfected with dual-luciferasereporter plasmids containing wild-type (WT) or mutant (Mut) ofTIMP3-3′UTR were treated with AngII (100 ng/ml), pre-miR-712 (20 nM) orcontrol pre-miR (20 nM) and anti-miR-712 (400 nM). Firefly luciferaseactivity (normalized to control Renilla luciferase) indicating TIMP3expression was determined using Luc-Pair miR Luciferase Assay Kit.

FIG. 10G shows data for RECK-3′UTR.

FIG. 10H shows data on frozen sections of abdominal aortas obtained fromAngII-infused C57BL/6 mice were used for immunofluorescence stainingwith antibody specific to TIMP3.

FIG. 10I shows data for antibody specific to TIMP3.

FIG. 10J shows in situ zymography where some abdominal aorta sectionswere incubated with the MMP inhibitor GM6001 (right bottom panel; scalebar=100 μm).

FIG. 10K shows data where iMAECs, pretreated with AngII (100 ng/ml)and/or premiR-712 (20 nM) for 1 day, were further treated withanti-miR-712 or mismatched control at 400 nM each as well as siRNAsagainst TIMP3 and RECK (siRECK or siTIMP3), respectively, at 100 nM eachfor 1 day. MMP activity was determined by cell-based ELISA usingDQ-gelatin.

FIG. 11A shows data where ApoE^(−/−) mice (24-26 weeks) were infusedwith AngII (1 μg/kg/min) or saline for 3 weeks via osmotic minipump andwere injected with mis-matched control (5 mg/kg) or anti-miR-712 (5mg/kg). ApoE^(−/−) mice (24-26 weeks) were infused with AngII (1μg/kg/min) or saline for 3 weeks via osmotic minipump and were injectedwith mis-matched control (5 mg/kg) or anti-miR-712 (5 mg/kg).Photographs showing macroscopic features of aneurysms induced by AngII.The square shows typical AAAs in ApoE^(−/−) mice. Lower panel showsrepresentative H&E staining for each group.

FIG. 11B shows data on the survival in anti-mR-712 treated groupcompared to saline or mis-matched anti-miR controls (n=10).

FIG. 11C shows data on incidence rate of AngII-induced AAA.

FIG. 11D shows data on the maximal abdominal aortic diameter quantitatedusing the H&E stained section; compared to saline or mis-match controlgroup. The data was analyzed using the Kruskal-Wallis test followed bythe Mann-Whitney U-test using Bonferroni correction to adjust theprobability (*p<0.05; n=10 each group).

FIG. 11E shows in situ zymography using DQ-gelatin to determine MMPactivity in AngII-infused abdominal aortic sections with saline,mis-matched control or anti-miR-712 injected group (scale bar=100 μm).

FIG. 11F shows systolic blood pressure measured in AngII-infused micetreated with anti-miR-712 compared to saline or mis-matched controls(n=10 each group).

FIG. 11G shows data where elastin fragmentation was evaluated byhistochemical staining with Orcein elastin stain kit using aortasections of saline, mis-matched control and anti-miR-712 treated groups.

FIG. 11H shows quantitation of elastin degradation. The data wasanalyzed using the Kruskal-Wallis test followed by the Mann-WhitneyU-test using Bonferroni correction to adjust the probability (*p<0.05;n=6 each group).

FIG. 12A shows data indicating injection of anti-miR-712 affects bothimmune cells and vessel wall cells, which in turn inhibit PBMCs adhesionto endothelium of suprarenal artery.

FIG. 12B shows data where abdominal aortic explants as well as PBMCswere obtained from vehicle or AngII-infused (1 μg/kg/min; 2 days)C57BL/6 mice that were also treated with mis-matched or anti-miR-712 (5mg/kg, s.c; 2 daily injections prior to AngII implantation). PBMCs(labeled with fluorescent Calcein) were then incubated for 30 min withthe abdominal aortic explants with their endothelial side up, and thePBMCs adhered to the endothelial surface was counted by confocalmicroscopy.

FIG. 12C shows data where macrophage (F4/80⁺) infiltration was examinedby the immunostaining the abdominal aorta section obtained in the frozensections described in FIG. 3D (scale bar, 100 μm; n=5 each group).

FIG. 13A shows data where TIMP3 expression was determined by qPCR iniMAECs pre-treated with premiR-205 (20 nM), anti-miR-205 (400 nM) ormis-matched control (400 nM) for 2 days, followed by AngII (100 ng/ml)treatment for another 1 day (n=4; *p<0.05; in triplicate).

FIG. 13B shows data for RECK.

FIG. 13C shows data where endothelial-enriched RNAs were prepared fromthe abdominal aortas of vehicle or AngII-infused mice, which werepre-treated with mis-matched control or anti-miR-205 (5 mg/kg; 2 dailyinjections), and miR-205 expression was determined by qPCR (n=4;*p<0.05; in triplicate). All data were analyzed using ANOVA followed byTukey's post hoc test, and values represent the mean±S.E.

FIG. 13D shows data for TIMP3.

FIG. 13E shows data for RECK.

FIG. 14A shows photographs showing macroscopic features of aneurysmsinduced by AngII. ApoE^(−/−) mice treated with anti-miR-205 ormis-matched control (5 mg/kg) were infused with AngII for 3 weeks. Thesquare shows typical AAAs in ApoE^(−/−) mice.

FIG. 14B shows data on the survival rate of AngII-induced AAA inanti-mR-205 treated group compared to mis-matched controls are shown.

FIG. 14C shows data on the incidence.

FIG. 14D shows data where maximal abdominal aortic diameter wasquantitated using the H&E stained section. The data was analyzed usingthe Kruskal-Wallis test followed by the Mann-Whitney U-test usingBonferroni correction to adjust the probability (*p<0.05 compared tomis-matched control, n=9 each group).

FIG. 14E shows in situ zymography using DQ-gelatin to determine MMPactivity in vehicle or AngII-infused abdominal aortic sections withmis-matched control or anti-miR-205 treated group (n=4, scale bar=100μm). Some sections from the AngII+mis-matched control treated mice wereincubated with GM6001 (upper right panel).

FIG. 14F shows systolic blood pressure measured in AngII-infused micetreated with anti-miR-205 compared to mis-matched controls (n=9).

FIG. 15 shows data indicating AngII-sensitive miRNAs expression in humanAAA. Expression of AngII-sensitive miRNA; miR-21, miR-1, miR-133b,miR-378 and human homolog of miR-712; miR-205 were determined by qPCRusing human normal aorta (n=4) and FFPE sections of human AAA wholetissues (n=5) obtained from Origene. (*p<0.05).

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, immunology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

“Subject” refers any animal, preferably a human patient, livestock,rodent, monkey or domestic pet.

The term “sample” as used herein refers to any biological or chemicalmixture for use in the method of the disclosure. The sample can be abiological sample. The biological samples are generally derived from apatient, preferably as a bodily fluid (such as tumor tissue, lymph node,sputum, blood, bone marrow, cerebrospinal fluid, phlegm, saliva, orurine) or cell lysate. The cell lysate can be prepared from a tissuesample (e.g. a tissue sample obtained by biopsy), for example, a tissuesample (e.g. a tissue sample obtained by biopsy), blood, cerebrospinalfluid, phlegm, saliva, urine, or the sample can be cell lysate. Inpreferred examples, the sample is one or more of blood, blood plasma,serum, cells, a cellular extract, a cellular aspirate, tissues, a tissuesample, or a tissue biopsy.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g., patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

Inhibition of Mechanosensitive microRNA, miR-712, Atypical microRNADerived from Pre-Ribosomal RNA, Decreases Endothelial Dysfunction andAtherosclerosis

Atherosclerosis typically occurs in arterial regions exposed todisturbed flow (d-flow) by mechanisms involving broad changes in geneexpression. While microRNAs (miRNAs) regulate various aspects ofcardiovascular biology and disease, their role in atherosclerosis hasnot previously been directly demonstrated. Vascular endothelial cellsrespond to blood flow through mechanosensors which transduce themechanical force associated with flow (known as shear stress) into cellsignaling events and changes in gene expression. D-flow and stable flow(s-flow) promotes and inhibits atherogenesis, respectively, in largepart by regulating discrete sets of pro-atherogenic and atheroprotectivegenes. While s-flow upregulates atheroprotective genes such as Klf2,Klf4, and eNOS, d-flow upregulates a number of pro-atherogenic genesincluding vascular cell adhesion molecule-1 (VCAM-1) and matrixmetalloproteinases (MMPs) which mediate pro-angiogenic,pro-inflammatory, proliferative, and pro-apoptotic responses, promotingatherosclerosis.

Using a mouse partial carotid ligation model and endothelial miRNA arraymechanosensitive miRNAs were identified. Of those mechanosensitivemiRNAs identified, miR-712 was the most shear-sensitive miRNAupregulated by d-flow both in vivo and in vitro. miR-712 is derived fromthe internal transcribed spacer 2 (ITS2) region of pre-ribosomal RNA(RN45S gene) in a XRN1 exonuclease-dependent, but DGCR8-independentmanner, suggesting that it is an atypical miRNA derived from anunexpected source. Studies including gain-of-function (pre-miR-712) andloss-of-function (anti-miR-712) approaches and target-binding assaysshowed that miR-712 directly downregulated the tissue inhibitor ofmetalloproteinase 3 (TIMP3) expression. This in turn activateddownstream metalloproteinases (MMPs and ADAM family) and stimulatedpro-atherogenic responses, endothelial tubule formation and sprouting,in a flow-dependent manner. Further, treatment with anti-miR-712prevented atherosclerosis in two different models of murineatherosclerosis using ApoE^(−/−) mice: a chronic conventionalwestern-diet or an acute carotid partial ligation model on a high-fatdiet. These results indicate that targeting mechanosensitive“athero-miRs” with anti-miR-based approaches is a viable treatmentparadigm in atherosclerosis.

Abdominal Aortic Aneurysm Induced by Angiotensin II is Mediated bymiR-712 and -205, which Target the Matrix Metalloproteinase Inhibitors,TIMP3 and RECK

The Angiotensin II (AngII)-induced abdominal aortic aneurysm (AAA) modelis commonly used rodent model that has been well studied because of manyfeatures it shares with human AAA. Uncontrolled degradation of the localextracellular matrix by proteases such as MMPs is an initiation step inAAA development. The regulation of protease activity by miRNA in AAA isreported herein. The miRNA-712 and -205 identified in this study in thevasculature are regulated by AngII and play a role in AngII-induced AAA.miR-712 was identified from a miRNA array and its human homolog miR-205was validated by endothelial-enriched RNAs obtained from C57BL6 miceinfused with AngII. Although it is not intended that embodiments of thedisclosure be limited by any particular mechanism, it is believed thatmiR-712 and miR-205 regulate matrix metalloproteases (MMPs) activitythrough directly targeting two inhibitors of MMPs: tissue inhibitor ofmetalloproteinase 3 (TIMP3) and reversion inducing cysteine-rich proteinwith kazal motifs (RECK). Silencing of miR-712 and miR-205 significantlydecreased MMP activity in the AngII-infused abdominal aorta wall,prevented dilatation of aorta and significantly reduced AAA incidence.AngII-sensitive miRNAs, miR-712 and miR-205, regulate MMP activitythrough TIMP3 and RECK and play a role in the pathogenesis of AAAindicating that targeting these miRNAs using their inhibitors is atherapeutic strategy to prevent the development of AAA and relateddiseases and conditions.

In Abdominal aortic aneurysm (AAA), a permanent dilation of theabdominal aorta occurs due to a loss of the structural integrity of thevascular wall. AAA is more common disease in subjects above the age of60 or 65. The most significant cause of mortality from AAA is acuterupture. The progressive weakening and dilation of the aorta observed inAAA is due to the degradation and remodeling of the extracellular matrix(ECM) of the aortic wall. Surgical and mechanical interventions are theonly known effective treatments to prevent AAA rupture.

AngII stimulates miR-712 and its human homolog, miR-205 expression inthe suprarenal artery, which in turn inhibits TIMP3 and RECK.Downregulation of these MMP endogenous inhibitors creates a permissiveenvironment for unregulated MMP activity in the abdominal aorta,therefore allowing the ECM remodeling leading to AAA. miR-712 isAngII-sensitive miRNA as determined through miRNA array, in situhybridization and qPCR validation in vitro and in vivo. miR-205 wasidentified as a human homolog of the murine miR-712. miR-712 and miR-205regulates metalloproteinase activity by targeting two endogenousinhibitors of MMPs and ADAMs; TIMP3 and RECK. Anti-miR-712 andanti-miR-205 treatment in vivo can effectively silence miR-712 andmiR-205 expression in arterial vessel wall cells and blood, restoringTIMP3 levels and inhibiting AAA development.

AAA are characterized by common molecular processes that underlieinflammation and ECM degradation. These changes are associated with aninflammatory infiltrate and excessive production of MMPs, which areassumed to organize the widespread matrix destruction. MMP degrade theECM proteins, whereas their inhibitors, TIMPs keep their activity incheck.

A mouse model to study the pathogenesis of AAA is AngII infusion modelusing osmotic mini-pump. In humans, there is associative evidencesupporting an increase in cardiovascular events with increases in theactivity of renin, the rate-limiting step in AngII generation and hasbeen shown to exert direct effects on vascular remodeling and function.The role of AngII in the atherogenic process has been inferred from thesurvival and ventricular enlargement trial that demonstrated thatadministration of angiotensin II-converting enzyme (ACE) inhibitors wasassociated with a decrease in cardiovascular morbidity and mortality. Anumber of animal experiments reported the relationship between AngII andaneurysm development and the preventive effect of ACE inhibitors andAngII receptor blocker (ARBs). Emerging evidence suggests that theAngII-induced AAA mouse model is a widely used approach to address thepathophysiological mechanism of this disease and rennin-angiotensinsystem may act as a molecular and therapeutic target for treating AAA.

Mechanisms of MMPs silencing include interaction with the specific TIMPsand other endogenous inhibitors, such as α2-macroglobulin, RECK andtissue-factor pathway-inhibitor 2 (TFPI2). TIMP3 and RECK wereidentified as target genes of miR-712 and miR-205. TIMPs inhibit abroader spectrum of metalloproteinases, however, not with the sameefficacy. Because of this broad inhibitory spectrum, TIMP-3 ablation inmice causes lung emphysema-like alveolar damage whereas TIMP-1-null miceand TIMP-2-null mice do not exhibit obvious abnormalities and loss ofTIMP3 promote AAA formation.

RECK was identified as another target gene of miR-712 and miR-205. RECKis a membrane bound protein which, in the mouse, has been found to beimportant in suppressing MMPs and angiogenesis in the metastaticcascade. miR-712 is a murine-specific miRNA. miR-205 shares the same“seed sequence” with miR-712 which are targets of TIMP3 and RECK.

Nucleobase Polymer Therapeutics

The term “nucleobase polymer” refers to a polymer comprising nitrogencontaining aromatic or heterocyclic bases that bind to naturallyoccurring nucleic acids through hydrogen bonding otherwise known as basepairing. A typical nucleobase polymer is a nucleic acid, RNA, DNA, orchemically modified form thereof. A nucleic acid may be single or doublestranded or both, e.g., they may contain overhangs. Nucleobase polymersmay contain naturally occurring or synthetically modified bases andbackbones. In certain embodiments, a nucleobase polymer need not beentirely complementary, e.g., may contain one or more insertions,deletions, or be in a hairpin structure provided that there issufficient selective binding.

In certain embodiments, the disclosure relates to composition comprisingan isolated antisense nucleobase polymers, interference nucleobasepolymers and RNA-blocking oligonucleotides.

In certain embodiments, the nucleobase polymers are 8 to 25 baseoligomers that mimic DNA or RNA. Many nucleobase polymers differ fromnative RNA or DNA in the chemical structure that links the four commonbases. For example, a RNA may be modified to contain phosphorothioatesinstead of phosphodiester linkages. Nucleobase polymers that containphosphorothioates may hybridize to RNA and promote RNase H mediateddegradation.

In certain embodiments, nucleobase polymers are contemplated to comprisepeptide nucleic acids (PNAs). One example of a peptide nucleic acid isone that has 2-aminoethyl glycine linkages or similar analogs in placeof the regular phosphodiester backbone. Other examples include d-lysPNA,argPNA, alternating units of 2-aminocyclopentanoic acid andpyrrolidine-2-carboxylic acid (pyrPNA). See Nielson, Chem &Biodiversity, 2010, 7:786.

In certain embodiments, nucleobase polymers are contemplated to comprisenon-natural nucleobases such as, but not limited to, pseudoisocytosineas a substitute for cytosine, diaminopurine as a substitute for adenine,bicyclic thymine analogue (7C1-bT), thiouracil, or combinations thereof.

In certain embodiments, nucleobase polymers are contemplated to comprisephosphorodiamidate morpholino oligomers (PMO). In certain embodiments,the nucleobase polymer comprises monomers of(2-(hydroxymethyl)morpholino)(piperazin-1-yl)phosphinate. In certainembodiments, the disclosure contemplates chemical conjugation of PMO toarginine-rich or cell penetrating peptides (CPP) such as (R-Ahx-R)₄(with Ahx standing for 6-aminohexanoyl), RXRRBRRXR ILFQY RXRBRXRB (SEQID NO: 6), RXRRBRRXR YQFLI RXRBRXRB (SEQ ID NO: 7),RXRRBRRX-ILFRY-RXRBRXRB (SEQ ID NO: 8), wherein X is 6-aminohexanoyl andB is β-alanine spacers. CPPs may be conjugated to the 3′ end of the PMOor to the 5′ end or both. See Warren & Bavari, Antiviral Research, 2012,94(1):80-88 and Betts et al., Molecular Therapy Nucleic Acids, 2012, 1:e38.

In certain embodiments, the disclosure relates to composition comprisingan isolated antisense nucleobase polymer that binds RNA of SEQ ID NO: 1or SEQ ID NO: 11. In certain embodiments, the nucleobase polymer is anucleic acid or nucleic acid mimetic that hybridizes to RNA of SEQ IDNO: 1 or SEQ ID NO: 11.

In certain embodiments, the antisense nucleobase polymer base comprisesmore than 10, 15, 20, 30, 40, 50, 60, or 70 nucleobases that basepair—hydrogen bond—with RNA of SEQ ID NO: 1 or SEQ ID NO: 11, or are thereverse complement of RNA of SEQ ID NO: 1 or SEQ ID NO: 11.

In certain embodiments, the nucleobase polymer base comprises one of thefollowing nucleobase sequences which are the miR663 reverse complementand segments:

(SEQ ID NO: 2) CCUUCCGGCGUCCCAGGCGGGGCGCCGCGGGACCGCCCUCGUGUCUGUGGCGGUGGGAUCCCGCGGCCGUGUUUUCCUGGUGGCCCGGCCAUG; (SEQ ID NO: 3)CCUUCCGGCGUCCCAGGCGGGGCGCCGCGGGACCGCCCUCGUGUCUGUGG C; (SEQ ID NO: 4)GGUGGGAUCCCGCGGCCGUGUUUUCCUGGUGGCCCGGCCAUG; (SEQ ID NO: 5)ACCGCCCUCGUGUCUGUGGCGGUGGGAUCCCGCGGC;

In certain embodiments, the nucleobase polymer base comprises one of thefollowing nucleobase sequences which are segments the miR205 reversecomplement

(SEQ ID NO: 12) CCGGTGGUUTGUUGGU; (SEQ ID NO: 13)TCCUCTGUUUTCTGGTTGGGTUTGUGU (SEQ ID NO: 14)  CUGUCTCCGGTGGUUTGUUGGU; and(SEQ ID NO: 15) CUGCTCCUTGCCTCCTGUUCTTCUCTCCUCTGUUUTCTGGTTGGG;

In certain embodiments, the disclosure relates to compounds,compositions, and methods useful for modulating of miR-205 and/ormiR-663 using nucleobase polymers. In particular, the instant disclosurefeatures small nucleic acid molecules, such as short interfering shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),and short hairpin RNA (shRNA) molecules and methods used to modulate theof miR-663.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering nucleobase polymers sometimes referred to aspost-transcriptional gene silencing or RNA silencing. The presence oflong dsRNAs in cells is thought to stimulate the activity of aribonuclease III enzyme referred to as Dicer. Dicer is thought to beinvolved in the processing of the dsRNA into short pieces of dsRNA knownas short interfering RNAs (siRNAs). Short interfering RNAs derived fromDicer activity are typically about 21 to about 23 nucleotides in lengthand comprise about 19 base pair duplexes. Dicer has also been implicatedin the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs)from precursor RNA of conserved structure that are implicated intranslational control. The RNAi response is thought to feature anendonuclease complex containing a siRNA, commonly referred to as anRNA-induced silencing complex (RISC), which mediates cleavage ofsingle-stranded RNA having sequence homologous to the siRNA. Cleavage ofthe target RNA takes place in the middle of the region complementary tothe guide sequence of the siRNA duplex. In addition, RNA interference isthought to involve small RNA (e.g., micro-RNA or miRNA) mediated genesilencing, presumably though cellular mechanisms that regulate chromatinstructure and thereby prevent transcription of target gene sequences. Assuch, siNA molecules can be used to mediate gene silencing viainteraction with RNA transcripts or alternately by interaction withparticular gene sequences, wherein such interaction results in genesilencing either at the transcriptional level or post-transcriptionallevel.

RNAi has been studied in a variety of systems. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Work in Drosophila embryonic lysateshas revealed certain preferences for siRNA length, structure, chemicalcomposition, and sequence that mediate efficient RNAi activity. Thesestudies have shown that 21 nucleotide siRNA duplexes are typical whenusing two 2-nucleotide 3′-terminal nucleotide overhangs. Substitution of3′-terminal siRNA nucleotides with deoxy nucleotides was shown to betolerated. Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is beneficial for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA. siRNA molecules lacking a 5′-phosphate are active whenintroduced exogenously.

A nucleobase polymer can be synthetic or recombinantly produced nucleicacid unmodified or chemically-modified compared to naturally occurringnucleic acids. A nucleic acid can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. Various chemically-modifiedsynthetic short interfering nucleic acid (siNA) molecules are capable ofmodulating miR-205 and/or miR-663 activity in cells by RNA interference(RNAi).

In one embodiment, the disclosure relates to a double-stranded shortinterfering nucleobase polymers that down-regulates miR-205 and/ormiR-663 or expression of miR-205 and/or miR-663, wherein said comprisingabout 15 to about 35 base pairs.

In certain embodiments, the nucleobase polymer or interference nucleicacid is in a hairpin.

In some embodiments, the disclosure relates to methods of treating asubject diagnosed with a vascular condition by administering apharmaceutical composition with a nucleobase polymer or nucleic acidthat is a single strand.

In certain embodiments, this disclosure relates to particles comprisinga hydrophilic or lipid membrane and ionizable or cationic corecomprising the nucleobase polymer. Siegwart et al. report the synthesisof coreshell nanoparticles by the reaction of epoxide-containing blockcopolymers with polyethylene glycol monomers and amines. See PNSA, 2011,108(32):12996-3001.

In certain embodiments, contemplated particles comprise block copolymersof poly(d,l-lactide) (PLA) or poly(d,l-lactide-co-glycolide) (PLGA) andpoly(ethylene glycol) (PEG), in which nucleobase polymers werephysically entrapped without chemical modification.

In certain embodiments, contemplated particles comprise a hydrophobicbiodegradable polymeric core that allows for the encapsulation andcontrolled release of nucleobase polymers, a hydrophilic shell thatprotects the nucleobase polymers, and optionally a targeting ligand thatmediated molecular interactions between particle and target endothelialcells.

In certain embodiments, contemplated particles comprise a linear polymerin which positively or negatively charged groups alternate withpolysaccharides (e.g., cyclodextrin). Upon mixing with nucleobasepolymers, the positively or negatively charged polymer respectivelyassociates with the negatively or positively charged backbone ofnucleobase polymers, nucleic acids, or RNAs. Several polymer/complexesself-assemble into a nanoparticle that fully protects the molecules fromdegradation in serum. Formation of inclusion complexes betweenadamantane (AD) and O-cyclodextrin allows noncovalent incorporation ofstabilizing (via PEG-AD conjugates) and/or targeting (via ligand-PEG-ADconjugates) components to a polymer-nucleic acid nanoparticles(polyplex). See Suzie & Davis, Bioconjugate Chemistry, 2002,13(3):630-639. Directly conjugating the nucleobase polymer to acyclodextrin-based polymer is also contemplated. See Heidel & Schluep,“Cyclodextrin-Containing Polymers: Versatile Platforms of Drug DeliveryMaterials,” J Drug Delivery, 2012, Article ID 262731, 17 pages.

In certain embodiments, the disclosure relates to a nucleobase polymersdisclosed herein optionally conjugated to a detectable marker or labelsuch as, but not limited to, a fluorescent dye, radio isotope, stableisotopes with lower natural abundance, positron-emitting radionuclide(tracer), antibody epitope, biotin, ligand, steroid, quantum dot. Usefulphysical properties include a characteristic electromagnetic spectralproperty such as emission or absorbance, magnetism, electron spinresonance, electrical capacitance, dielectric constant or electricalconductivity. The marker may be ferromagnetic, paramagnetic,diamagnetic, luminescent, electrochemiluminescent, fluorescent,phosphorescent, chromatic or have a distinctive mass. Fluorescentmoieties which are useful as markers include dansyl fluorophores,coumarins and coumarin derivatives, fluorescent acridinium moieties andbenzopyrene based fluorophores and quantum dots. In general, theseproperties are based on the interaction and response of the marker toelectromagnetic fields and radiation and include absorption in the UV,visible and infrared regions of the electromagnetic spectrum, presenceof chromophores which are Raman active, and can be further enhanced byresonance Raman spectroscopy, electron spin resonance activity, positronemission tomography, and nuclear magnetic resonances and use of a massspectrometer to detect presence of a marker with a specific molecularmass.

Synthesis of Nucleobases Polymers

Small nucleobase polymers and nucleic acid motifs (“small” refers tonucleic acid motifs no more than 100 nucleotides in length, preferablyno more than 80 nucleotides in length, and most preferably no more than50 nucleotides in length; e.g., individual oligonucleotide sequences orsequences synthesized in tandem) are preferably used for exogenousdelivery. Exemplary molecules of the instant disclosure are chemicallysynthesized, and others can similarly be synthesized.

One synthesizes oligonucleotides (e.g., certain modifiedoligonucleotides or portions of oligonucleotides) using protocols knownin the art as, for example, described in U.S. Pat. No. 6,001,311. Thesynthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-endand phosphoramidites at the 3′-end. In a non-limiting example, smallscale syntheses are conducted on a 394 Applied Biosystems, Inc.synthesizer using a 0.2 micro mol scale protocol with a 2.5 min couplingstep for 2′-O-methylated nucleotides and a 45 second coupling step for2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Alternatively,syntheses at the 0.2 micro mol scale can be performed on a 96-well platesynthesizer, such as the instrument produced by Protogene (Palo Alto,Calif.) with minimal modification to the cycle. A 33-fold excess of2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazolecan be used in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 22-fold excess of deoxy phosphoramidite anda 70-fold excess of S-ethyl tetrazole mop can be used in each couplingcycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Otheroligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc.synthesizer include the following: detritylation solution is 3% TCA inmethylene chloride (ABI); capping is performed with 16% N-methylimidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF(ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water inTHF (PerSeptive Biosystems, Inc.). S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65 degrees for 10 minutes. After cooling to −20degrees, the supernatant is removed from the polymer support. Thesupport is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1,vortexed and the supernatant is then added to the first supernatant. Thecombined supernatants, containing the oligonucleotide, are dried.

Alternatively, the nucleic acid molecules can be synthesized separatelyand joined together post-synthetically, for example, by ligation or byhybridization following synthesis and/or deprotection.

-   -   Nucleic acids can also be assembled from two distinct nucleic        acid strands or fragments wherein one fragment includes the        sense region and the second fragment includes the antisense        region of the RNA molecule.

The nucleic acid molecules can be modified extensively to enhancestability by modification with nuclease resistant groups, for example,2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H). Constructs can bepurified by gel electrophoresis using general methods or can be purifiedby high pressure liquid chromatography and re-suspended in water.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency. See e.g., U.S. Pat.Nos. 5,652,094, 5,334,711, and 6,300,074. All of the above referencesdescribe various chemical modifications that can be made to the base,phosphate and/or sugar moieties of the nucleic acid molecules describedherein. Modifications that enhance their efficacy in cells, and removalof bases from nucleic acid molecules to shorten oligonucleotidesynthesis times and reduce chemical requirements are desired.

In one embodiment, nucleic acid molecules include one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. AG-clamp is a tricyclic aminoethyl-phenoxazine 2′-deoxycytidine oranalogue. See Lin &. Matteucci, J Am Chem Soc, 1998, 120, 8531-8532;Flanagan, et al., Proc Nat Acad Sci USA, 1999, 96, 3513-3518; and Maier,et al., Biochemistry, 2002, 41, 1323-1327. A single G-clamp analogsubstitution within an oligonucleotide can result in substantiallyenhanced helical thermal stability and mismatch discrimination whenhybridized to complementary oligonucleotides. The inclusion of suchnucleotides in nucleic acid molecules results in both enhanced affinityand specificity to nucleic acid targets, complementary sequences, ortemplate strands.

In another embodiment, nucleic acid molecules include one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid”nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see forexample U.S. Pat. Nos. 6,639,059, 6,670,461, 7,053,207).

In another embodiment, the disclosure features conjugates and/orcomplexes of nucleobase polymers. Such conjugates and/or complexes canbe used to facilitate delivery of polymers into a biological system,such as a cell. Contemplated conjugates include those with cellpenetrating peptide. The conjugates and complexes provided may imparttherapeutic activity by transferring therapeutic compounds acrosscellular membranes, altering the pharmacokinetics, and/or modulating thelocalization of nucleic acid molecules. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system, with or without degradable linkers. Thesecompounds improve delivery and/or localization of nucleic acid moleculesinto a number of cell types originating from different tissues, in thepresence or absence of serum (see U.S. Pat. No. 5,854,038). Conjugatesof the molecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

In yet another embodiment, nucleobase polymers having chemicalmodifications that maintain or enhance enzymatic activity of proteinsinvolved in RNAi are provided. Such nucleic acids are also generallymore resistant to nucleases than unmodified nucleic acids. Thus, invitro and/or in vivo the activity should not be significantly lowered.

In another aspect a nucleobase polymers comprises one or more 5′ and/ora 3′-cap structure, for example on only the sense strand, the antisensestrand, or both strands.

A “cap structure” refers to chemical modifications, which have beenincorporated at either terminus of the oligonucleotide. See, forexample, Adamic et al., U.S. Pat. No. 5,998,203. These terminalmodifications protect the nucleic acid molecule from exonucleasedegradation, and may help in delivery and/or localization within a cell.The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal(3′-cap) or may be present on both termini. In non-limiting examples,the 5′-cap includes, but is not limited to, glyceryl, inverted deoxyabasic residue (moiety); 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non-bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925).

In one embodiment, the disclosure features modified nucleobase polymer,with phosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkyl silyl, substitutions.

Pharmaceutical Compositions

The following protocols can be utilized for the delivery of nucleobasepolymers. A nucleobase polymer can be adapted for use to prevent ortreat a vascular disease or condition that is related to or will respondto the levels of miR-205 and/or miR-663 in the blood, a cell, or tissue,alone or in combination with other therapies. For example, a nucleobasepolymer can be contained in a delivery vehicle, including liposomes, foradministration to a subject, carriers and diluents and their salts,and/or can be present in pharmaceutically acceptable formulations. U.S.Pat. Nos. 6,395,713 and 5,616,490 further describe general methods fordelivery of nucleic acid molecules. Nucleobase polymers can beadministered to cells by a variety of methods known to those of skill inthe art, including, but not restricted to, encapsulation in liposomes,by iontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for example U.S.Pat. Nos. 7,141,540 and 7,060,498), poly(lactic-co-glycolic)acid (PLGA)and PLCA microspheres (see for example U.S. Pat. No. 6,447,796),biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (U.S. Pat. No. 7,067,632). In another embodiment,the nucleobase polymers can also be formulated or complexed withpolyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives.

In one embodiment, a nucleobase polymers is complexed with membranedisruptive agents such as those described in U.S. Pat. No. 6,835,393. Inanother embodiment, the membrane disruptive agent or agents andnucleobase polymers are also complexed with a cationic lipid or helperlipid molecule, such as those lipids described in U.S. Pat. No.6,235,310.

Embodiments of the disclosure feature a pharmaceutical compositioncomprising one or more nucleobase polymers in an acceptable carrier,such as a stabilizer, buffer, and the like. The nucleobase polymers oroligonucleotides can be administered (e.g., RNA, DNA or protein) andintroduced into a subject by any standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. When it is desired to use a liposome delivery mechanism,standard protocols for formation of liposomes can be followed. Thecompositions can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for administration byinjection, and the other compositions known in the art.

Embodiments of the disclosure also feature the use of the compositioncomprising surface-modified liposomes containing poly (ethylene glycol)lipids (PEG-modified, or long-circulating liposomes or stealthliposomes). These formulations offer a method for increasing thecirculation and accumulation of in target tissues. The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA. See U.S. Pat. Nos. 5,820,873 and 5,753,613. Long-circulatingliposomes are also likely to protect from nuclease degradation.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Methods of Use

In certain embodiments, the disclosure relates to methods of treating orpreventing a vascular disease or condition comprising administering aneffective amount of a pharmaceutical composition disclosed herein to asubject in need thereof.

In certain embodiments, the disclosure relates to methods of treating orpreventing a vascular disease condition comprising administering aneffective amount of a pharmaceutical composition comprising annucleobase polymer or RNAi method disclosed herein or a particlecomprising RNAi composition disclosed herein.

In certain embodiments, the disclosure relates to methods for inhibitingor preventing atherosclerosis, aortic aneurysm, rheumatoid arthritis andcancer in human patients by treating them with using an effective amountof a compound that inhibits miR-205/miR-712/miR-663 family, which inturn increases or preserves expression of tissue inhibitor ofmetalloproteinease-3 (TIMP3) and reversion-inducing cysteine-richprotein with Kazal motifs (RECK), wherein said compound is selected fromthe group consisting of microRNA inhibitors of miR-712/miR-205/miR-663family by using nucleic acid sequences that complement the whole or apart of the miRNA sequence such as the seeding sequence. The nucleicacid inhibitors can be synthesized by using locked nucleic acid(LNA)-based designs.

In certain embodiments, the disclosure relates to methods comprisingadministering an effective amount of a microRNA-712/miR-205/miR-663antagonist to the diseased tissues and cell types.

In certain embodiments, the subject is a human that is at risk of,exhibiting symptoms of, or diagnosed with atherosclerosis, peripheralvascular disease, coronary heart disease, heart failure, rightventricular hypertrophy, cardiac dysrhythmia, endocarditis, inflammatorycardiomegaly, myocarditis, vascular heart disease, stroke,cerebrovascular disease, or peripheral arterial disease.

In certain embodiments, the subject has type I or type II diabetes,impaired glucose tolerance, elevated serum C-reactive proteinconcentration, vitamin B6 deficiency, dietary iodine deficiency,hypothyroidism, hyperlipidemia, hypertension, or is older than 50 yearsold, or smokes cigarettes daily.

In certain embodiments, the pharmaceutical composition is administeredin combination with a statin, atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,simvastatin, ezetimibe, amlodipine, niacin, aspirin, omega-3 fatty acid,or combinations thereof.

In certain embodiments, the therapy is delivered by intravenous orintramyocardial injection. In certain embodiments, the intramyocardialinjection is delivered during surgery. In certain embodiments, theintramyocardial injection is delivered percutaneously using one or morecatheters. In certain embodiments, the intramyocardial injection isdirectly into the epicardium. In certain embodiments, the therapy isdelivered by intrapericardial injection. In certain embodiments, themethod of delivery is combined with one or more vasodilatory agents.

In certain embodiments, one or more of the above therapies isadministered in combination with one or more other therapies forcardiovascular disease.

In certain embodiments, the disclosure relates to medical devices, suchas stents, e.g., mesh tube comprising compositions disclosed herein,e.g., recombinant TIMP3, antisense nucleobase polymers, blockingoligonucleotide, gene therapy vectors, and RNA interference therapeuticcompositions.

In certain embodiments, the disclosure relates to using compositionsdisclosed herein coated or conjugated or integrated into vascular ornon-vascular medical devices, stents, pace makers, guide wires, deliveryballoons, catheters, bioresorbable vascular scaffolds, embolicprotection devices and others. Molecular compositions can be linked topolymers on the surface of the devices or integrated to release withbiodegradable polymers.

In certain embodiments, the disclosure relates to a gene therapy using avector that expresses human TIMP3 in human cells. In certainembodiments, the disclosure relates to methods for inhibiting orpreventing atherosclerosis, aortic aneurysm, rheumatoid arthritis andcancer in human patients by treating them with using an effective amounta vector that expresses TIMP3 in human cells. In some embodiments, thedisclosure relates to methods of treating a subject diagnosed with avascular condition by administering an effective amount a vector thatexpresses TIMP3 in human cells.

In certain embodiments, the disclosure relates to gene therapy. The genetherapy typically comprises a recombinant viral vector. In certainembodiments, the viral vector is a recombinant retrovirus. In certainembodiments, the retroviral vector is a recombinant lentivirus. Incertain embodiments, the viral vector is a recombinant adenovirus. Incertain embodiments, the viral vector is a recombinant adeno-associatedvirus (rAAV) of any rAAV serotype.

In certain embodiments, the gene therapy comprises a non-viral vector.In certain embodiments, the non-viral vector is plasmid DNA. In certainembodiments, the non-viral vector is a polymer-DNA complex. In certainembodiments, the non-viral vector is a liposome-DNA complex.

Recombinant viral vectors include, but are not limited to, recombinantadeno-associated virus (rAAV), recombinant retrovirus, recombinantadenovirus, recombinant poxvirus, and recombinant herpes simplex virus(HSV). The choice of recombinant viral vector used depends on the typeof genetic material (e.g. RNA or DNA, single-stranded ordouble-stranded) to be transferred to the target cell. Several types ofretrovirus are available including, but not limited to, lentivirus andgamma-retrovirus (e.g. murine leukemia virus), with lentivirus being thetypical choice. A retrovirus may be used to transfer RNA, which issubsequently incorporated randomly into the target cell's genome by theenzymes reverse transcriptase and integrase. Multiple serotypes of rAAV(e.g. AAV1, AAV2, and other AAV serotypes) may be used. rAAV may be usedto transfer single-stranded DNA. Recombinant adenovirus does notincorporate into a target cell's genome, but rather expresses free DNAin the nucleus and may be considered transient. Recombinant adenovirusmay be used to transfer double-stranded DNA. Poxvirus is used totransfer double-stranded DNA. HSV is typically used to target neurons asit is capable of latently infecting those cells. HSV may be used totransfer double-stranded DNA. Of the viral vector methods of genetherapy, rAAV-mediated gene delivery is the typical choice. rAAV isconsidered non-immunogenic, i.e. an individual will not typically mountan immune response to clear it, and it is capable of infectingnon-dividing, quiescent cells.

In certain embodiments, the disclosure relates to 10, 15 or 20 or morenucleotide segments of microRNA-712/miR-205/miR-663 that are expressedby a vector to form small hairpin RNA. In certain embodiments, thedisclosure relates to vectors that express a small hairpin RNA whereinthe double stranded portion contains a 10, 15 or 20 or more nucleotidesegment of microRNA-712/miR-205/miR-663. RNA molecules may be used tointerfere with gene expression. Small hairpin RNA (shRNA), also known asshort hairpin RNA, may be introduced into a target cell and may beconstitutively expressed by the H1 or U6 promoters. The shRNA is capableof inhibiting target gene expression by RNA interference. shRNA iscleaved, forming small interfering RNA (siRNA). siRNAs aredouble-stranded. The RNA-induced silencing complex binds to the siRNA,and the siRNA in turn binds to a target mRNA sequence which is thencleaved. This may be used to disrupt mRNA translation. Antisense RNA mayalso be used to modulate mRNA translation. Antisense RNA issingle-stranded RNA that binds to complementary mRNA thereby obstructingits ability to translate.

Gene therapy is currently a well-characterized, established methodologywith both viral and non-viral delivery well-known in the art. Forexample, clinical trial were conducted to restore the levels of thesarcoplasmic reticulum Ca²⁺⁻ATPase enzyme in patients with advancedheart failure by gene transfer with the use of a viral vector (AAV1) todeliver the SERCA2a gene, demonstrating safety and feasibility. SeeRapti et al., Can J Cardiol., 2011; 27(3):265-83. See also Herzog etal., “Two decades of Clinical gene therapy—success is finally mounting”Discov Med. 2010; 9(45):105-11. Other examples of diseases in whichclinical trials are being conducted by use of gene therapies include α1antitrypsin, Batten's disease, Canavan's disease, Cystic fibrosis,Haemophilia B, Leber's congenital amaurosis, Pompe's disease, Musculardystrophy, Parkinson's disease, Age-related macular degeneration, andRheumatoid arthritis. See Mingozzi & High, Nat Rev Genet. 2011,12(5):341-55.

One ordinarily skilled in the art will be capable of creating andadministering a recombinant viral vector for the purpose of genetherapy. A recombinant viral vector may be created by a processcomprising isolating the transgene of interest, incorporating it into arecombinant viral expression vector (the construct), and administeringthe construct to the target in order to incorporate the transgene.Numerous recombinant viral expression vectors are commercially availablefor these purposes. The exact protocol for generating a recombinantviral vector may vary depending on the choice of the viral vector.Example protocols for generating rAAV, the typical choice of viralvector, as well as recombinant retrovirus are disclosed in Heilbronn R,Weger S., Viral vectors for gene transfer: current status of genetherapeutics. Handb Exp Pharmacol. 2010, (197):143-70.

In certain embodiments, the disclosure relates to the treatment orprevention of an inflammatory disorder comprising compositions disclosedherein in an effective amount to a subject in need thereof. Contemplatedinflammatory disorders include, but not limited to, acne vulgaris,asthma, celiac disease, chronic prostatitis, glomerulonephritis,hypersensitivities, inflammatory bowel disease, pelvic inflammatorydisease, reperfusion injury, rheumatoid arthritis, sarcoidosis,transplant rejection, vasculitis, interstitial cystitis,atherosclerosis, allergies, hay fever, myopathies, leukocyte defects, avitamin A deficiency and autoimmune diseases.

Examples

miR-712 was the Most Consistently and Robustly Upregulated miRNA Both InVivo and In Vitro

Vascular endothelial cells respond to blood flow through mechanosensorswhich transduce the mechanical force associated with flow (known asshear stress) into cell signaling events and changes in gene expression.D-flow and stable flow (s-flow) promotes and inhibits atherogenesis,respectively, in large part by regulating discrete sets ofpro-atherogenic and atheroprotective genes. While s-flow upregulatesatheroprotective genes such as Klf2, Klf4, and eNOS, d-flow upregulatesa number of pro-atherogenic genes including vascular cell adhesionmolecule-1 (VCAM-1) and matrix metalloproteinases (MMPs) which mediatepro-angiogenic, pro-inflammatory, proliferative, and pro-apoptoticresponses, promoting atherosclerosis.

A mouse model of flow-induced atherosclerosis by partially ligating theleft carotid artery (LCA) of the ApoE^(−/−) mouse is reported in Nam etal., American journal of physiology Heart and circulatory physiology,2009, 297(4): H1535-1543. This model directly demonstrates the causalrelationship between d-flow and atherosclerosis as the LCA rapidlydevelops robust atherosclerosis within two weeks following partialligation that causes d-flow with characteristic low and oscillatoryshear stress (OS), while the contralateral, undisturbed right commoncarotid artery (RCA) remains healthy. In addition, a method of isolatingintimal RNA from mouse carotid artery following ligation has beendeveloped. This method allows easy and rapid endothelial-enriched RNAisolation that is virtually free of contamination from the vascularsmooth muscle cells and leukocytes. Using this mouse model and the RNAisolation method, more than 500 mechanosensitive genes were discovered,including ones such as lmo4, klk10, and dhh, and confirming well-knownones, such as VCAM-1, klf2 and eNOS5. However, the effect of d-flow onendothelial miRNAs, especially in vivo, has not been clear. To identifymechanosensitive miRNAs in arterial endothelium in vivo, endothelialmiRNA array analyses was carried out using the mouse partial carotidligation model and integrated these two genome-wide data-sets using asystems biology approach to help us identify relevant miRNA-target geneconnection.

The in vivo microarray data showed that 45 (27 up- and 18 downregulated)miRNAs were altered by more than 50% in the LCA endothelium compared tothe RCA at 48 hours post-ligation. Quantitative PCR (qPCR) usingadditional independent RNA samples was used to validate the miRNA arraydata for the top 10 most mechanosensitive miRNAs (5 up-, 5down-regulated miRNAs at 48 hours post-ligation): upregulated (miR-330*,712, 699, 223 and 770-5p) and down-regulated (miR-195, 30c, 29b, 26b andlet-7d) miRNAs. To determine whether these mechanosensitive miRNAs thatwere identified in vivo responded specifically to shear stress,expression of these miRNAs was tested in vitro using immortalized mouseaortic endothelial cells (iMAECs) that were subjected to laminar (LS) oroscillatory stress (OS), mimicking s-flow and d-flow in vivo,respectively. These results showed that miR-712 was the mostconsistently and robustly upregulated miRNA both in vivo and under flowconditions in vitro, leading us to further characterize its expressionand function.

In Situ Hybridization with a miR-712 Probe Showed SignificantlyIncreased Expression of miR-712 in the LCA Endothelium

A time-course study showed that miR-712 was upregulated significantly inthe d-flow region (LCA) compared to s-flow region (RCA) at 48 hpost-ligation in mouse carotids. Using the miScript qPCR assay,pre-miR-712 expression significantly increased at 48 h post-ligationwhile expression of mature-miR-712 increased significantly in LCA at 24and 48 h post-partial ligation, which was subsequently validated bysequencing the PCR amplicons. This suggests that initial processing ofpre-miR-712 into the mature form precedes (24h) the active synthesis ofpre-miR-712 (48h) in mouse artery in vivo. Exposure of mouse aorticendothelial cells (iMAECs) to oscillatory shear stress for 24 h in vitroalso dramatically increased expression of mature and precursor forms ofmiR-712 compared to laminar shear, confirming the flow-sensitivity ofmiR-712 both in vivo and in vitro. Studies using in situ hybridizationwith a miR-712 probe (Exiqon) showed significantly increased expressionof miR-712 in the LCA endothelium, but not in control RCA, furthervalidating the microarray and qPCR results. To determine whetherincreased expression of miR-712 in LCA was not simply due to an acutechange in mechanical environment created by the partial ligationsurgery, we tested expression of miR-712 in mouse aortic arch, where theatherosclerosis-prone lesser curvature (LC) and athero-protected greatercurvature (GC) are naturally and chronically exposed to d-flow ands-flow, respectively. Expression of miR-712 was significantly higher inthe flow-disturbed LC compared to GC. These findings suggest thatmiR-712 is indeed a flow-sensitive miRNA, which is significantlyupregulated in endothelial cells exposed to d-flow in the aortic archnaturally or in LCA upon partial carotid ligation, or in vitro.

Ribosomal RNAs a Source of miRNAs and siRNA Treatment Increased theExpression of miR-712

The genomic locus and biogenesis pathway of miR-712 were not known. Anucleotide sequence search revealed that the pre-miR-712 sequencematched to the internal transcribed spacer region 2 (ITS2) region of themurine pre-ribosomal RNA, RN45s gene, which is still not completelyannotated. The Rn45S encodes for 45S, which contains sequences for 18S,5.8S and 28S rRNAs along with two intervening spacers, ITS1 and ITS2.Since ribosomal RNAs have never been reported to be a source of miRNAs,whether miR-712 biogenesis was regulated by the canonical pathway usingthe DGCR8/DROSHA and DICER microRNA processors or by non-canonicalpathways in a flow-dependent manner was examined. Expression of DICER1and DGCR8 was not regulated by flow in endothelial cells in vitro(iMAEC) and in vivo (mouse carotids and aortic arch). Since, XRN1 is anexonuclease that is known to degrade the ITS2 region during theprocessing of RN45s, whether XRN1 is shear-dependent and regulatesbiogenesis of miR-712 was tested. It was found that XRN1 expression wasdownregulated by d-flow in the mouse carotid and aortic arch and inendothelial cells in vitro. Further, knockdown of XRN1 by siRNAtreatment increased the expression of miR-712, suggesting that reductionof XRN1 under d-flow conditions led to accumulation of miR-712. Incontrast, knockdown of the canonical miRNA processor DGCR8 did notaffect miR-712 expression. These results suggest that miR-712 is amurine-specific, non-canonical miRNA derived from an unexpected source,pre-ribosomal RNA, in a XRN1-dependent, but DGCR8-independent andDICER1-dependent manner.

Murine-miR-712 and Human miR-663 Serve as Pro-Atherogenic miRNAs

To test whether this novel mechanism of miRNA biogenesis frompre-ribosomal RNA exists in humans as well, we examined human RN45S genefor putative miRNAs using a computational predication program, miREval.This search revealed that human-specific miR-663 could be derived fromRN45s gene, in addition to its previously annotated genomic locus onchromosome 2 (miRBase). Interestingly, like miR-712, miR-663 waspreviously identified from a microarray study as one of the mostshear-sensitive miRNAs in human endothelial cells and was shown toinduce endothelial inflammation, a key atherogenic step, raising thepossibility that they share a common biogenesis pathway and roles inendothelial function. Consistent with this hypothesis, we found thatXRN1 expression is inhibited by oscillatory shear stress and that siRNAknockdown of XRN1 significantly increased miR-663 expression in humanendothelial cells. These results indicate that murine-miR-712 and humanmiR-663 could be derived from respective RN45s genes by a commonmechanism involving XRN1 and serve as pro-atherogenic miRNAs.

miR-712 is Responsible for Metalloproteinase-Dependent MatrixDysregulation in Endothelium in a TIMP3-Dependent Manner

Through in silico analysis using miR-712 predicted target gene list fromTargetScan and the mechanosensitive gene list using the same mousemodel, TIMP3 was identified as a potential gene target. TIMP3 wasselected for further study since the miR-712 interactome analysissuggested its potential role as a key gene hub connected to numerousgenes of interest, including matrix metalloproteinases-2/9 (MMP2/9) anda disintegrin and metalloproteinase 10 (ADAM10), a disintegrin andmetalloproteinase with thrombospondin type 1 motif, 4 (ADAMTS4) andversican that are known to play a role in the regulation ofpathophysiological angiogenesis and atherosclerosis.

To test whether miR-712 directly targets TIMP3 in endothelial cell, adual luciferase reporter assay was employed in iMAECs transfected witheither wild-type or mutated TIMP3 3′-UTR firefly luciferase constructs.Treatment of cells with pre-miR-712 inhibited luciferase activity in adose-dependent manner, while mutant or control pre-miR had no effect,demonstrating that miR-712 directly binds TIMP3 3′-UTR and inhibits itsexpression in endothelial cells.

Whether TIMP3 expression was regulated by flow in a miR-712-dependentmanner was tested. Exposure of iMAECs to oscillatory shear for 24 hsignificantly decreased TIMP3 mRNA and protein expression, compared tolaminar shear. Pre-miR-712 treatment decreased expression of TIMP3 inlaminar shear-exposed cells, while inhibition of miR-712 using lockednucleic acid (LNA)-based anti-miR-712 increased TIMP3 expression incells exposed to oscillatory shear. These findings demonstrate thatmiR-712 upregulated by oscillatory shear, is directly responsible forthe loss of TIMP3 expression in endothelial cells.

Inhibition of miR-712 by Anti-miR-712 Treatment or Overexpression ofTIMP3 can Overcome Endothelial Dysfunction

To examine whether d-flow-induced miR-712 was responsible formetalloproteinase-dependent matrix dysregulation in endothelium in aTIMP3-dependent manner, endothelial tubule formation and sprouting wasstudied as functional markers of extracellular matrix change in vascularpathophysiology. Endothelial tubule formation was stimulated by exposingcells to oscillatory shear compared to laminar shear. This oscillatoryshear-induced endothelial tubule formation was inhibited by anti-miR-712treatment, while pre-miR-712 treatment increased tubule formation inlaminar shear stressed cells. Further, the tubule formation induced bypre-miR-712 treatment was completely prevented by TIMP3 overexpression,while siRNA-mediated knockdown of TIMP3 robustly stimulated tubuleformation with or without pre-miR-712. Similarly, endothelial sproutingwas stimulated by miR-712 overexpression, which was abrogated by TIMP3overexpression. These results indicate that mechanistically TIMP3 isdownstream of miR-712 and inhibition of miR-712 by anti-miR-712treatment or overexpression of TIMP3 can overcome endothelialdysfunction induced by d-flow in a miR-712/TIMP3 dependent manner.

LNA-Based Anti-miR-712 Regulates TIMP3 Expression In Vivo

Next, to test whether d-flow-induced miR-712 plays a key role inatherosclerosis, mice were treated with LNA-based anti-miR-712. It wasfound that anti-miRs can be effectively delivered to arterialendothelium in vivo as demonstrated by two independent methods: confocalen face imaging of TexasRed-labeled control LNA-anti-miR andautoradiography of 64Cu-labelled anti-miR-712. Interestingly, bothfluorescent-labeled anti-miR and radiolabelled anti-miR preferentiallyaccumulated in the flow-disturbed LCA and aortic arch, which is likelydue to increased endothelial mass transfer and permeability tomacromolecules in d-flow areas. A dose-curve study showed thatanti-miR-712 (5 mg/kg dose, injected via s.c.) effectively inhibitedd-flow-induced miR-712 expression in LCA endothelium as compared to themismatched control, and this dose was used for all subsequent studies.We found that expression of TIMP3 mRNA and protein was reduced in d-flowregions both in the partially ligated LCA and aortic arch LC compared toRCA and GC, respectively, and it was rescued by the anti-miR-712treatment. This further demonstrates the role of miR-712 in regulationof TIMP3 expression in a flow-dependent manner in vivo. Since TIMP3 is awell-known inhibitor of MMPs and ADAMs, the effect of anti-miR-712 wasexamined on these metalloproteinase activities in vivo. MMP activity (insitu zymography) and ADAMs activity (versican cleavage byimmunohistochemical staining) were increased by d-flow in LCA comparedto RCA which were significantly blunted by anti-miR-712 treatment. Thesein vivo results, taken together with the endothelial tubule formationand sprouting results in vitro, provide strong evidence for a directhierarchal link between d-flow, miR-712, TIMP3, and its downstreammetalloproteinase activity in arterial wall.

Inhibitory Effect of Anti-miR-712 in Two Different Murine Models ofAtherosclerosis

The role of miR-712 in atherosclerosis was tested in two differentmodels of atherosclerosis using ApoE^(−/−) mice: (1) the partial carotidligation model fed a high-fat diet that rapidly develops atherosclerosiswithin 2 weeks 5 and (2) the conventional western diet-fed model thatchronically develops atherosclerosis in 3 months 45. In the carotidligation model, the LCA rapidly developed atherosclerosis within 2 weeksand treatment with anti-miR-712 (5 mg/kg/day, s.c. twice a week for 2weeks) significantly reduced atherosclerosis lesion development andimmune cell infiltration compared to the mismatched-anti-miR-712 orsaline controls. Similarly, anti-miR-712 treatment (5 mg/kg/day, s.c.twice a week for 3 months) significantly inhibited atherosclerosisdevelopment in the conventional western diet model in whole aortas aswell as in the aortic arch. In both models, anti-miR-712 did not affectthe serum lipid profile. These findings demonstrate a potent inhibitoryeffect of anti-miR-712 in two different murine models ofatherosclerosis.

These results demonstrate the hierarchal link between d-flow, miR-712,TIMP3, and its downstream metalloproteinase to endothelial dysfunctionand atherosclerosis. Although it is not intended that embodiments ofthis disclosure be limited by any particular mechanism, it is believedthat d-flow sensed by mechanosensors in endothelium reduces theexpression of XRN1, which increases miR-712 expression derived from thepre-ribosomal RNA. It is believed that increased miR-712 downregulatesTIMP3 expression, which in turn activates MMPs and ADAM proteases,leading to arterial wall matrix fragmentation, endothelial dysfunctionand atherosclerosis. Targeting these metalloproteinase regulators byanti-miRs may serve as a therapeutic approach to treat atherosclerosis.

LNA-Based Anti-miR Inhibitor Treatment In Vivo.

For in vivo delivery of anti-miR, inhibition of miR-712 in artery, andfunctional test of miR-712 on atherosclerosis, locked nucleic acid(LNA)-based miRNA inhibitor (anti-miR-712, Exiqon) were subcutaneously(s.c.) administered to mice. To test delivery efficiency of anti-miRinto mouse arterial endothelium, C57BL/6 mice (n=3) were subcutaneouslyadministrated with 5 mg/kg dose of Texas-Red-615-labeledanti-miR-control (TEX615/ACGTCTATACGCCCA) (SEQ ID NO: 9) or saline.After 24 hour post injection, animals were sacrificed by CO₂ inhalationand perfusion with saline containing heparin, followed by a secondperfusion with 10% formalin. Aortas were carefully excised, dissectedfree of surrounding fat tissues. The tissue samples from LCA and RCA aswell as the aortic arch were then mounted on glass slides using aqueousfluorescence mounting medium (Dako) after counterstaining with4′,6-diamidino-2-phenylindole (DAPI, Sigma). Samples were imaged using aZeiss LSM 510 META confocal microscope (Carl Zeiss). To study theinhibition of miR-712 in in vivo settings, LNA-based anti-miR-712(GTACCGCCCGGGTGAAGGA) (SEQ ID NO: 10) or mismatched control wassubcutaneously administered to C57BL/6 mice (n=4) in an increasing doseof 5, 20 and 40 mg/kg on the day before partial ligation surgery and onthe day of partial ligation surgery. Animals were sacrificed on the 4day post ligation and the carotid intimal RNAs were extracted andexpression of miR-712 was determined by qPCR. For the functional testingof miR-712 on atherosclerosis, anti-miR-712 or mismatched-control wassubcutaneously administered (5 mg/kg) to ApoE−/− mice (n=10) at one daybefore partial ligation surgery, on the day of partial ligation surgeryand every third day thereafter for 2 weeks. Mice were fed HFD for entireduration of the experiment. Aortic trees were dissected out andprocessed for subsequent plaque analysis and immunohistochemicalanalysis.

Synthesis of anti-miR712-DOTA.

The conjugation of 5′-modified anti-miR-712 (5′-/5AmMC6/+CGCCCGGGTGAAGA(SEQ ID NO: 16)-3′, Exiqon, and DOTA-NHS-ester(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester), Macrocyclics) followed a Hnatowich et al.,Journal of pharmacology and experimental therapeutics, 1996, 276(1):326-334, with modifications. 5′-modified anti-miR-712 (0.19 μmol, 1 mg)was suspended in 0.2 M borated buffer (0.3 mL, pH 8.5) and thedimethylsulfoxide solution (20 μL, Aldrich) of DOTA-NHS-ester (3.6 μmol,2.8 mg) was added. After 5 hours of reaction at room temperature, themixture of DOTA conjugated anti-miR-712 was purified on Sephadex G-25filled with double distilled water (GE healthcare, exclusion limit >5 k)to remove excess residual DOTA-NHS-ester. Isolated anti-miR-712-DOTA wasthen purified by HPLC monitored by absorbance (260 nm), with thefollowing HPLC conditions: column Clarity Oligo-PR (250×10.00 mm, 5micron, Phenomenex), gradient-solvent B (5-30%, 0-30 min, solvent A-0.1M triethylammonium acetate (TEAA), solvent B-acetonitrile). Collectedfractions were concentrated by centrifugation with an Amicon Ultra-4centrifugal filter unit (MWCO: 3 k). Double distilled water (4 mL) wasadded to the filter unit and the mixture was concentrated again bycentrifugation. The washing procedure was repeated 6 times to removetriethylammonium acetate salt. The concentrated solution was lyophilizedand anti-miR-712-DOTA was stored at −20° C. MS analysis of sample wasperformed with Thermo Fisher Scientific LTQ Orbitrap (San Jose, Calif.)operated in the centroid mode. The deconvoluted mass from 1413.43 (Z=4)was detected at 5657.7 (Calc MW=5657.6)

Radiolabeling of Anti-miR-712-DOTA

⁶⁴CuCl2 was purchased from MIR Radiological Sciences (WashingtonUniversity Medical School, St. Louis, Mo.) under a protocol controlledby the University of California, Davis. ⁶⁴CuCl2 (3-5 mCi) was added into0.1 M ammonium citrate buffer (0.1 mL, pH 5.5) and the anti-miR-712-DOTA(1 mM, 3-4 μL) in double distilled water was slowly added. Afterincubation for 1 hour at 40° C., the pH was adjusted to 7 by 1 M sodiumhydroxide solution (trace metal, Aldrich). The reaction mixture wasaspirated into 5 mL TEAA (10 mM, pH 7.0-7.5) in a 10 mL syringe. Thesolution was slowly eluted through a Sep-Pak light C18 cartridge(Waters, Milford, Mass.) prewashed by methanol and 10 mM TEAA. The C18cartridge was washed with 10 mM TEAA (4 mL) two times and eluted by 95%methanol (0.5-0.8 mL) to release the radiolabeled product into a 2-mLtube. The solvent was evaporated to dryness with a gentle stream ofnitrogen gas at 40° C. Isolated ⁶⁴Cu-labeled anti-miR-712-DOTA wasdissolved in a phosphate buffered-saline (0.2 mL). Concentrated solutionwas diluted with PBS to prepare a dose of ˜0.2-0.3 mCi/150 μL, which wasfiltered through 0.2 μm of a Whatman Anotop syringe filter (Dassel,Germany). The specific activity of ⁶⁴Cu-anti-miR-712-DOTA was ˜500-750mCi/μmol. Radiochemical purity measured by a binary pump HPLC system(Waters), was more than 95%.

Isolation of Endothelial-Enriched RNA from the LC and GC Regions of theMouse Aortic Arch

Ascending aortas were harvested from C57BL/6 mice and opened en face.The endogenous d-flow region (lesser curvature; LC) and s-flow regions(greater curvature; GC) were identified and carefully dissected out. Theendothelium was placed against a nitrocellulose-membrane soaked inisopropanol for 5 minutes, and the media and adventitia were peeled awayleaving the endothelial monolayer adherent to the nitrocellulosemembrane. The nitrocellulose membrane was then used to extract the miRNAusing Qiagen miREasy kit. The left over adventitia samples werevisualized under microscope to ensure that the endothelial layer wasproperly transferred onto the nitrocellulose membrane. For eachexperiment, LC and GC regions from two to four mice were pooled. A panelof markers genes for endothelium (PECAM1), smooth muscle (aSMA) andimmune cell (CD11b) was used to determine the enrichment of endothelialRNA from the preparation.

Ang II Induced miR-712 Expression In Vivo and In Vitro

A time-dependent miR-712 expression was tested in AngII infusedabdominal aortic endothelium as well as the leftover sample (containingRNAs of medial smooth muscle cells and adventitial cells). AngIIinfusion increased miR-712 expression between 12-72 h time-points inboth the endothelial and media/adventitia samples (FIG. 9a ). AngIItreatment induced miR-712 expression in both iMAECs and VSMCs in vitro(FIG. 9b ). Next, in situ hybridization was performed to furthervalidate the AngII-sensitivity of miR-712 expression in the abdominalaortic endothelium. Studies using in situ hybridization with a miR-712probe (Exiqon) showed a robust expression of miR-712 in the cytoplasm(arrows) and nuclei of the abdominal aortic endothelium, compared to thevehicle (FIG. 9c ). These results suggest that AngII treatment increasesmiR-712 expression both in endothelial cells and smooth muscle cells inthe mouse abdominal aorta in vivo as well as in vitro.

TIMP3 and RECK are Direct Targets of miR-712

Through in silico analysis using TargetScan, an additional potentialtarget of miR-712, RECK was identified in response to the humoral AngIIstimulation. Since TIMP3 and RECK are well-known negative regulators ofMMP activity, a critical player in AAA development and progression, weexamined whether miR-712 indeed targeted TIMP3 and RECK expression usinggain-of-function (premiR-712) and loss-of-function (anti-miR-712)approaches in the AngII-dependent manner. Treatment with premiR-712 andAngII significantly decreased TIMP3 and RECK mRNA expression, both ofwhich were blocked by anti-miR-712 treatment in iMAEC (FIGS. 10a and 10b) in vitro. In addition, AngII-stimulated miR-712 induction as well asdownregulation of TIMP3 and RECK were significantly reversed in micetreated with anti-miR-712 (FIGS. 10d and 10e ). For this study,anti-miR-712 was subcutaneously injected twice (1 and 2 days prior toAngII implantation) at 5 mg/kg/day dose, and effectively silencedAngII-induced miR-712 expression (FIG. 10c ).

To further determine whether miR-712 bound to and inhibited TIMP3 andRECK expression directly in an AngII-dependent manner, we performed theluciferase assay, in which a construct containing the 3′-UTR region ofTIMP3 or RECK mRNA containing the putative miR-712 binding sequence wasused. Treatment of iMAECs with premiR-712 and AngII inhibited luciferaseactivity of TIMP3 and RECK, while their mutants or control-premiR showedno effect (FIGS. 10f and 10g ). In addition, anti-miR-712 blocked theinhibitory effect of premiR-712 or AngII on TIMP3 and RECK luciferaseactivity (FIGS. 10f and 10g ). Together, these data suggest that TIMP3and RECK are direct targets of miR-712 in response to AngII.

Whether AngII downregulates TIMP3 and RECK expression by amiR-712-dependent mechanism in vivo was tested by immunostaining.Expression of TIMP3 and RECK were evident in endothelial and smoothmuscle cells in the vehicle control groups (FIGS. 10h and 10i ). AngIIinfusion decreased the expression of TIMP3 and RECK compared to thevehicle, but anti-miR-712 treatment reversed it (FIGS. 10h and 10i ).Since TIMP3 and RECK are well-known inhibitors of MMPs, we examined theeffect of anti-miR-712 on MMP activity in vivo by using an in situzymography assay using the fluorescent DQ-gelatin. As shown in FIG. 10j, AngII infusion increased MMP activity as evidenced by the greenfluorescent signal intensity, but was prevented by treating mice withanti-miR-712 in vivo or the MMP inhibitor GM6001 added during thezymography assay. This in situ zymography result was further confirmedin an in vitro cell-based assay using iMAEC (FIG. 10k ). The in vitrostudy showed that AngII induced MMP activity, which was prevented byanti-miR-712 treatment.

Whether TIMP3 or RECK, or both were important player in regulation ofthe AngII-dependent MMP activity was investigated. For this study, cellspre-treated with AngII and anti-miR-712 were treated with siRNAs toknockdown TIMP3, RECK, or both. The inhibitory anti-miR-712 effect onthe MMP activity was partially blunted when cells were treated withTIMP3 siRNA or RECK siRNA (FIG. 10k ). Interestingly, knockdown of bothTIMP3 and RECK together did not produce the additive effect, which maybe due to an insensitive assay condition or an unknown cooperationbetween the two inhibitors. Together, these results demonstrate thatAngII stimulates MMP activity by inducing expression of miR-712, whichin turn downregulates TIMP3 and RECK, both of which seem to be equallyimportant in MMP activity regulation.

Anti-miR-712 Inhibits AAA Induced by AngII Infusion

Whether anti-miR-712 can prevent AAA induced by AngII infusion inApoE^(−/−) mice was tested using the Daugherty method. For this study,mice were treated with anti-miR-712 using the same dosage and protocolused above in FIG. 10c-e , except for additional anti-miR-712 injectionsevery 4 days for 3 weeks following AngII implantation. AngII infusion (1μg/kg/min) for 3 weeks induced pronounced AAA phenotype in thesuprarenal region of the abdominal aorta, which was significantlyblunted in the anti-miR-712-treated mice compared to the mis-matched orsaline-treated control groups (FIG. 11a ). While 20% (2 out of 10 miceeach) of AngII-infused mice died during the first week in the saline andmis-matched groups, none of the anti-miR-712 treated mice showedmortality for the duration of the study (FIG. 11b ). Also, treatmentwith anti-miR-712 dramatically reduced AAA incidence to 20% (2 of 10mice) compared to the mis-matched (80%; 8/10) or saline controls (70%;7/10) (FIG. 11c ). Similarly, the increase in AngII-induced abdominalaortic diameter was also significantly reduced by the anti-miR-712treatment compared to the saline and mis-matched controls (FIG. 11d ).

Whether anti-miR-712 could inhibit AngII-induced MMP activity was testedby the in situ zymography using DQ-gelatin. The MMP activity (shown asthe intense fluorescent gelatin signal) was dramatically higher inAngII-infused mice (AngII+saline group) compared to the vehicle control,but it was remarkably reduced in the anti-miR-712-treated mice comparedto the mis-matched control (FIG. 11e ).

Whether the anti-AAA effect of anti-miR-712 was mediated throughnormalizing blood pressure was testeom in the AngII-infused mice.AngII-infusion significantly increased blood pressure within one weekfollowing the AngII infusion, but anti-miR-712 treatment did not alterthe blood pressure (FIG. 11f ). Aortic wall elastin fragmentation is animportant feature of AAA in both humans and mouse models. The elasticlamina degradation were graded on a scale of 1 (least) to 4 (worst) inour mouse samples. The elastic laminas were frequently disrupted andfragmented in AngII-infused mice treated with saline or mis-matchedcontrols, whereas anti-miR-712 treated mice showed little signs ofelastin fragmentation (FIGS. 11g and 11h ). These results suggest thatthe anti-AAA effect of anti-miR-712 is mediated in an MMP-dependentmanner, but is independent of the pressor response.

Anti-miR-712 Inhibits Both Endothelial and Circulating LeukocyteInflammation

Given the importance of inflammation in AAA development, whether theeffect of anti-miR-712 (delivered systemically via s.c. injection) wasmediated through the aortic endothelial cells, circulating leukocytes orboth was examined. To test this hypothesis, an ex vivo leukocyteadhesion assay was performed using abdominal aorta explants andperipheral blood monocytic cells (PBMCs) obtained from the mice treatedwith anti-miR-712 or mis-matched control along with vehicle orAngII-infusion. Three groups of aortas were obtained from the mice thatwere treated with 1) vehicle, 2) AngII+mis-matched control, or 3)AngII+anti-miR-712. In addition, PBMCs were also obtained from the samethree groups of mice as above. These 3 groups of aortic explants and 3groups of PBMCs were then used in a 3×3 combination study (FIG. 12a ).Here, PBMCs were added to an aortic explant with its endothelial surfaceup in a dish, and the number of PBMCs adhering to the endothelialsurface after a 30 min incubation time was microscopically quantitated.First, adhesion of vehicle-control PBMCs to the vehicle-controlendothelial surface was very low (FIG. 12a , panel 1). Second, bothPBMCs and aortic explants obtained from AngII-infused mice showedsignificantly increased adhesion as compared to the vehicle control (asindicated by the increased number of PBMCs shown as green dots) (FIG.11a : panel 1 vs. 2; panel 1 vs. 4). Third, both the aorta explants andPBMCs obtained from anti-miR-712 treated mice showed a significantreduction in PBMC adhesion to endothelium compared to the mis-matchedcontrols (FIG. 11a : panel 3 vs. 2; panel 7 vs. 4), suggesting theanti-inflammatory effect of anti-miR-712 treatment. The quantitativeresults shown in FIG. 4B further supported these points. Consistent withthese ex vivo results, we also found a robust F4/80⁺ monocyte/macrophagestaining in AngII-infused mice with saline or mis-matched controls, butit became nearly undetectable in the anti-miR-712 treated mice (FIG. 11c). These findings suggest that the anti-AAA effect of anti-miR-712 ismediated, at least in part, by inhibiting inflammation of both aorticendothelial cells and circulating leukocytes.

The miR-205, a Human Homolog of miR-712, Directly Targets TIMP3 and RECK

Since miR-712 is murine-specific, its clinical implication for humandisease could be limited. To address this potential concern, itspotential human homolog, miR-205, was identified which shares the 7-mer“seed sequence” with miR-712 and is highly conserved in most mammalianspecies (TargetScan) including mouse and human. Whether miR-205 alsotargets TIMP3 and RECK in endothelial cells in an AngII-dependent mannerby using a gain-of-function (pre-miR-205) and loss-of-function(anti-miR-205) approaches was tested. Treatment of iMAEC with premiR-205or AngII decreased TIMP3 and RECK mRNA expression, which was preventedby anti-miR-205 treatment (FIGS. 13a and 13b ). Similarly, AngIItreatment increased expression of miR-205 in mouse aortic endotheliumand the media+adventitial cells in vivo (FIG. 13c ). AngII-inducedmiR-205 expression was effectively silenced by anti-miR-205 treatment inmice (FIG. 13c ), using the same dosage and injection protocol used foranti-miR-712 (FIG. 13c-e ). AngII treatment decreased TIMP3 and RECKexpression, which was reversed by anti-miR-205 treatment in mouse aorticendothelium (FIGS. 13d and 13e ). These results suggest that miR-205expression is AngII-sensitive and it targets TIMP3 and RECK, likemiR-712.

Treatment with Anti-miR-205 Prevents Ang II-Induced AAA

To determine whether miR-205 plays an important role in AAA,AngII-infused ApoE^(−/−) mice were treated with anti-miR-205 ormis-matched control using the same protocol as anti-miR-712. As shown inFIG. 14a-d , anti-miR-205 treatment significantly decreased AAAincidence, mortality, and abdominal aorta dilation compared to themis-matched control. While the survival rate of AngII-infused andmis-matched group was 56% (5/9), the anti-miR-205-treated group showed88.8% survival rate at the 3 week time-point (FIG. 14b ). Anti-miR-205treatment significantly reduced AAA incidence (2/9; 22.2%) compared tothe mis-matched group (7/9; 77.7%) (FIG. 14c ). Next, the effect ofanti-miR-205 treatment on MMP activity was tested in the same groups ofmice by in situ zymography. AngII-induced MMP activity was nearlyblocked by treating the aorta section of the AngII-treated mice with theMMP inhibitor GM6001 during the in situ zymography assay (FIG. 14e ;upper right panel). Anti-miR-205 treatment dramatically reduced the MMPactivity compared to the mis-matched control (FIG. 14e ). These resultssuggest that the AngII- and miR-205-sensitive metalloproteinase activityis mostly accounted for by the MMP activity. Like anti-miR-712,anti-miR-205 also did not affect AngII-induced systemic hypertension inthese mice (FIG. 14f ). Taken together, these results demonstrate that,like anti-miR-712, anti-miR-205 treatment has a potent preventive effecton AAA induced by AngII infusion.

AngII-Sensitive miRNAs in the Murine AAA are Also Upregulated in HumanAAA Tissues

Whether some of the AngII-sensitive miRNAs identified in this mousestudy, including miR-205, -21, -1, 133b and 378 were also upregulated inhuman AAA tissues was tested. For this study, total RNAs prepared fromde-identified human AAA paraffin sections (n=5) were compared to thosewithout the disease (non-AAA, n=4) from Origene. miR-205 expression was˜2-fold higher in AAA samples compared to the non-AAA (FIG. 15). Also,expression of miR-21, miR-133b, and miR-378, but not miR-1, wassignificantly higher in human AAA samples compared to the non-AAA. Thisresult suggests that miRNAs, especially the human homolog of miR-712,miR-205, identified in our AngII-induced murine AAA model appears to berelevant in human AAA as well.

Anti-miR-205 in the Treatment of Atherosclerosis

Doses (e.g., 200 mg of aqueous solution pH adjustment to 7.5-8.5) of thenucleotide anti-miR205 GCCTCCTGAACTTCACTCCA (SEQ ID NO: 17) asG*-mC*-mC*-T*-mC*-dmC-dT-dG-dA-dA-dmC-dT-dT-dmC-dA-mC*-T*-mC*-mC*-A*[d=2′-deoxy,*=2′-O-(2-methoxyethyl), mC 5-methylcytidine] are administered byinjection repeatedly to a subject. In certain embodiments, it iscontemplated that any reverse complement sequence of a twenty nucleotidewindow of human miR-205 can be used to substitute the nucleotides in theabove sequence provided C is 5-methylcytidine.

The invention claimed is:
 1. A method of treating or preventing avascular disease selected from abdominal aortic aneurysm comprisingadministering an effective amount of a pharmaceutical compositioncomprising a single stranded nucleobase polymer to a subject in needthereof, wherein the single stranded nucleobase polymer binds miR-205(SEQ ID NO: 11) AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA wherein the nucleobase polymer bindssufficiently to prevent translation of tissue inhibitor ofmetalloproteinase 3 in vivo.
 2. The method of claim 1 wherein thenucleobase polymer comprises GCCTCCTGAACTTCACTCCA (SEQ ID NO: 17). 3.The method of claim 1 wherein the nucleobase polymer comprisesCCGGTGGUUTGUUGGU (SEQ ID NO: 12).
 4. The method of claim 1 wherein thenucleobase polymer comprises monomers of phosphodiester,phosphorothioate, methylphosphonate, phosphorodiamidate, piperazinephosphorodiamidate, ribose, 2′-O-methy ribose, 2′-O-methoxyethyl ribose,2′-fluororibose, deoxyribose,1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol,P-(2-(hydroxymethyl)morpholino)-N,N-dimethylphosphonamidate,morpholin-2-ylmethanol, (2-(hydroxymethyl)morpholino)(piperazin-1-yl)phosphinate, or peptide nucleic acids and combinationsthereof.
 5. The method of claim 1 wherein the nucleobase polymer is 3′or 5′ terminally conjugated to a hydrocarbon, polyethylene glycol,saccharide, polysaccharide, cell penetrating peptide, or combinationsthereof.
 6. The method of claim 5 wherein the cells penetrating peptideis a positively charged peptide, arginine-rich peptide, or oligoargininepeptide.
 7. The method of claim 6 wherein the oligoarginine peptide isocta-arginine.
 8. The method of claim 1, wherein the subject is a human.9. The method of claim 1, wherein the pharmaceutical composition isadministered in combination with a statin, atorvastatin, cerivastatin,fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,rosuvastatin, simvastatin, ezetimibe, amlodipine, niacin, aspirin,omega-3 fatty acid, or combinations thereof.
 10. A method of treating orpreventing atherosclerosis comprising administering an effective amountof a pharmaceutical composition comprising a single stranded nucleobasepolymer to a subject in need thereof, wherein the single strandednucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGA GGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 11. A method oftreating or preventing peripheral vascular disease comprisingadministering an effective amount of a pharmaceutical compositioncomprising a single stranded nucleobase polymer to a subject in needthereof, wherein the single stranded nucleobase polymer binds miR-205(SEQ ID NO: 11) AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA wherein the nucleobase polymer bindssufficiently to prevent translation of tissue inhibitor ofmetalloproteinase 3 in vivo.
 12. A method of treating or preventingcoronary heart disease comprising administering an effective amount of apharmaceutical composition comprising a single stranded nucleobasepolymer to a subject in need thereof, wherein the single strandednucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 13. A method oftreating or preventing heart failure comprising administering aneffective amount of a pharmaceutical composition comprising a singlestranded nucleobase polymer to a subject in need thereof, wherein thesingle stranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA whereinthe nucleobase polymer binds sufficiently to prevent translation oftissue inhibitor of metalloproteinase 3 in vivo.
 14. A method oftreating or preventing right ventricular hypertrophy comprisingadministering an effective amount of a pharmaceutical compositioncomprising a single stranded nucleobase polymer to a subject in needthereof, wherein the single stranded nucleobase polymer binds miR-205(SEQ ID NO: 11) AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 15. A method oftreating or preventing cardiac dysrhythmia comprising administering aneffective amount of a pharmaceutical composition comprising a singlestranded nucleobase polymer to a subject in need thereof, wherein thesingle stranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 16. A method oftreating or preventing endocarditis comprising administering aneffective amount of a pharmaceutical composition comprising a singlestranded nucleobase polymer to a subject in need thereof, wherein thesingle stranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA wherein the nucleobase polymerbinds sufficiently to prevent translation of tissue inhibitor ofmetalloproteinase 3 in vivo.
 17. A method of treating or preventinginflammatory cardiomegaly comprising administering an effective amountof a pharmaceutical composition comprising a single stranded nucleobasepolymer to a subject in need thereof, wherein the single strandednucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 18. A method oftreating or preventing myocarditis comprising administering an effectiveamount of a pharmaceutical composition comprising a single strandednucleobase polymer to a subject in need thereof, wherein the singlestranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 19. A method oftreating or preventing vascular heart disease comprising administeringan effective amount of a pharmaceutical composition comprising a singlestranded nucleobase polymer to a subject in need thereof, wherein thesingle stranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 20. A method oftreating or preventing stroke comprising administering an effectiveamount of a pharmaceutical composition comprising a single strandednucleobase polymer to a subject in need thereof, wherein the singlestranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.
 21. A method oftreating or preventing cerebrovascular disease comprising administeringan effective amount of a pharmaceutical composition comprising a singlestranded nucleobase polymer to a subject in need thereof, wherein thesingle stranded nucleobase polymer binds miR-205 (SEQ ID NO: 11)AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACAwherein the nucleobase polymer binds sufficiently to prevent translationof tissue inhibitor of metalloproteinase 3 in vivo.